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Cellular expression of murine leukemia virus gp70-related antigen on thymocytes of uninfected mice correlates with Fv-4 gene-controlled resistance to friend leukemia virus infection
VIROLOGY
128, 127-139
(1983)
Cellular Expression of Murine Leukemia Virus gp70-Related Antigen Thymocytes of Uninfected Mice Correlates with Fv-4 Gene-Controlled Resistance to Friend Leukemia Virus Infection HIDETOSHI Department
IKEDA’
of Genetics, Institute 4-6-l Shirokanedai, Received
August
AND of Medical Minabku,
TAKESHI
ODAKA of
Science, University Tokyo 108, Japan
25, 1982; accepted
March
on
Tokyo,
29, 1983
Alleles at the Fv-4 locus have been shown to determine susceptibility (Fv-P) and resistance (Fv-4’) to infection with ecotropic murine leukemia viruses (MuLV). The resistance is dominant in heterozygous mice. BALB/c mice are Fv-4” and a semicongenic line BALB/c-Fv-hw’, homozygous for Fv-4’ allele and called C4W, is being developed. Extracts of ‘l-labeled thymocytes from BALB/c and C4W mice were precipitated by a series of antisera against MuLV and the precipitates examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Thymocytes from C4W mice expressed 80K and 73K glycoproteins related to the gp70 of MuLV, while only the 73K glycoprotein was occasionally detectable on BALB/c thymocytes. Genetic studies showed that a locus controlling expression of the 80K protein was dominant and associated with the resistant allele at Fv-4. This membrane gp70 may be important in the Fv-h-mediated resistance to exogenous virus infection. INTRODUCTION
eluding FLV and the expression of endogenous ecotropic MuLV are almost completely restricted (Kai et aZ., 1976; Odaka et al., 1980; Gardner et al., 1980). Recently we established a partially congenie mouse line BALB/c-Fv-4~’ (C4W) which carries the Fv-4’ allele from a wild mouse on the BALB/c background (Odada et ah, 1981). During the course of establishing the congenic line, we immunized BALB/c mice repeatedly with lymphoid cells from progeny mice derived from crosses between BALB/c and BALB/c-FV..$w~‘, in an attempt to identify the gene product specified by the Fv-4’ allele. Some of the pooled antisera were found to recognize a cell surface alloantigen associated with the Fv-4’ allele (Yoshikura and Odaka, 1982). In this study we describe the characterization of this cell surface antigen. The results indicate that at least two MuLV gp70-related molecules are expressed on the plasma membranes of thymocytes of C4W mice, one of which appears unique to previously described membrane gp7Os (Old and Stockert, 1977; Tung et al., 1976). The
The Fv-4 locus controls susceptibility to Friend leukemia virus (FLV) infection and also influences sensitivity to a variety of ecotropic murine leukemia viruses (MuLVs) (Suzuki, 19’75; Suzuki and Matsubara, 1975; Kai et al., 1976; Yoshikura et ab, 19’79; Odaka et ah, 1981). This locus is located on chromosome 12 (Ikeda et al., 1981) and has two alleles, Fv-4” (susceptibility) and Fv-4’ (resistance). The dominant resistance allele was found in one strain of laboratory mice (Suzuki, 1975) and in many wild Asian mice (Odaka et al., 1978), and is presumably identical to the Akvr-1 locus described by Gardner et al. (1980) in wild California mice. In Fv-4’ mice and Akvr-1’ mice, the replication of exogenously infected ecotropic MuLVs in’ Present Address: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 20205. Author to whom requests for reprints should be addressed. 127
0042-6822183 Copyright All rights
$3.00
CC 1983 by Academic Press, Inc. of reproduction in any form reserved
128
IKEDA
AND
identification of such a cell surface gp70 molecule provides a possible explanation for Fv-&associated resistance. MATERIALS
AND
METHODS
Mice. BALB/c, AKR/J,and C57BL/6(B6) mice were bred in our laboratory. FRG mice, previously called G mice (Suzuki, 1975; Suzuki and Matsubara, 1975), and A/ J mice were provided by Dr. S. Suzuki, Research Laboratories, Chugai Pharmaceutical Company, Ltd., Tokyo, Japan. NZB/ San mice were obtained from the Laboratory of Experimental Animals of our Institute. 129/Sv-SZCp(129) mice were a kind gift from Dr. T. Noguchi, National Institute of Genetics, Mishima, Japan. The partially congenic mouse line BALB/c-Fv-QW’ (C4W) was established by introducing the Fv-4’ gene from a wild mouse onto the BALB/c (Odaka et al, 1981). Mice used here were at the sixth backcross generation. C57BL/6-Fv” is a congenic strain carrying the Fv-2” allele of DDD mice on the C57BL/ 6 background (Odaka and Matsukura, 1969). BALB.D is a congenic strain carrying the H-Z complex of DDD mice on the BALB/c background (Ikeda, unpublished data). All the mice used for testing thymocyte antigens were 3 to 12 weeks old and of both sexes. Antisera. The antisera used in this work are listed in Table 1. In this report, we describe two different types of specificity in the three Fv-4 alloantisera (anti-Fv4wTs,anti-G, and anti-C4W sera). One precipitated gp70-related cell surface antigens on both C4W and AKR thymocytes, while the other defined a gp70-related thymocyte antigen which is unique to Fv-4’ mice. We tentatively term these “groupspecific antisera” and “Fv-4 type-specific antisera,” respectively (see Table 2). The specificity of anti-FLV serum was indistinguishable from that of the group-specific antiserum. By each recipient-donor combination (BALB/c anti-Fv-4’“, (BALB/ c X DDD)Fi anti-G, and BALB/c antiC4W), both group-specific and Fv-4 typespecific antisera were obtained. However, to avoid confusion, we used a limited number of antisera lots throughout this work;
ODAKA
anti-Fv-4w’” (Lots 85 and 1121) and antiC4W (Lots 41ml and 41m4) as Fv-4 typespecific antisera, and anti-G (Lot 1210) as group-specific antiserum. All of these antisera were used without special absorption. We used a panel of five antisera in experiments of Table 2. The reactivities of these sera for MuLV antigens were tested (Ikeda and Odaka, manuscript in preparation). The “Fv-4 type-specific” antisera precipitated none of viral proteins tested. The “group-specific” and anti-FLV sera precipitated gp7Os from all ecotropic and some dualtropic MuLV strains but no xenotropic MuLVs. The anti-gp70 and antiRLV sera precipitated gp7Os from all ecotropic, xenotropic, and dualtropic MuLV strains tested. P15(E)s from all ecotropic, dualtropic, and some xenotropic MuLV strains were precipitated by “group-specific,” anti-FLV, and anti-RLV sera, and weekly by anti-gp70. Most gag proteins of all MuLV strains tested were precipitated only by anti-RLV. Virus and virus assay. NB-tropic Friend leukemia virus (FLV) that had been serially passaged in BALB/c mice was prepared from the spleen homogenate of virus-infected BALB/c mice. The lymphatic leukemia virus (LLV) titer was measured on C-182 cells (Bassin et al, 1971), using the XC-plaque assay (Rowe et aL, 1970). The titer was expressed as PFU/spleen. Linkage analysis of cell surface antigens on the thywwcytes with the Fv-4 genotype. A mouse of C4W line, which was heterozygous for Fv-4 and at the ninth backcross generation, was backcrossed to a BALB/c mouse. The hybrid mice were thymectomized at about 4 weeks of age. Thymocytes were used for the surface iodination. Ten days after thymectomy mice were inoculated intraperitoneally with 0.2 ml of NBtropic FLV (1.5 X lo3 FFU and 1.9 X lo5 PFU). Individual spleens were weighed 10 days later and homogenates titered for LLV. Cell surface iodinaticm. Thymocytes suspended at a concentration of l-2.5 X 10’ per ml in 0.1 or 0.2 ml phosphate-buffered saline (PBS) were iodinated by incubation with 50 fig of lactoperoxidase (Boehringer
h-6
GENE
AND
CELL TABLE
MEMBRANE
gp70
129
1
ANTISERA USED Abbreviation Anti-Fv-4w”
Anti-G Anti-C4W Anti-H-2 Anti-FLV Anti-RLV
Immunization
and descriptions
BALB/c mice were immunized with normal spleen, thymus, and lymph node cells from Fv-4” and Fv-4” mice which were born during the course to establish the congeneic C4W mice, Some of the pooled sera contain cytotoxic antibody to a cell surface antigen linked to the Fv-4’ allele (H. Yoshikura and T. Odaka, 1982). (BALB/c X DDD)F, mice were immunized with spleen cells from G (FRG) mice with Fv4” genotype. BALB/c mice were immunized with spleen cells and thymocytes from C4W mice. BALB.D mice were immunized with spleen cells from BALB/c mice. This alloantiserum contained antibodies to H-zd antigens as revealed by hemagglutination test and a low titer of antibody against gp70 of MuLV (data not shown). C57BL/6-Fv” mice were inoculated with N-tropic FLV, developed, and then recovered from splenomegaly. These mice were bled and the sera pooled. This antiserum precipitates gp70 and p15(E) of FLV (Dr. K. Kai, personal communication). A rabbit was immunized with purified RLV produced by JLS-V9 cells (Wright et al, 1967). This was a kind gift from Dr. K. Kai.
Anti-MuLV,
A rabbit was immunized with SiRC cells,a rabbit cell line, infected with xenotropic MuLV (Yoshida and Yoshikura, 1981), as described by Morse et al (1979). This was a kind gift from Dr. H. Yoshikura.
Anti-gp70 Anti-p30 Anti-p15 Anti-p12 Anti-p10
Goats were immunized with gp70, ~30, ~15, ~12, or p10 purified from RLV. These were kind gifts of The Division of Cancer Cause and Prevention, NCI.
Mannhaim) and 0.5 mCi of Na’%I (New for formalin-fixed Staphylococcus aweus England Nuclear) at room temperature. at 4” for 60-120 min, and immune comThe reaction was initiated by the addition plexes were collected by centrifugation. of HaOa. Ten microliters of 0.03% of HaOa Precipitates were washed three times with was sequentially added at 2- to 5-min in- PBS containing 0.5% sodium deoxychotervals and the reaction terminated by late, 0.1% sodium dodecyl sulfate (SDS), washing the cells with RPM1 1640 medium and 1% Triton X-100, or NET buffer concontaining 5% precolostrum newborn calf taining 0.05% NP-40,150 mMNaC1,50 mM serum followed by two washes with PBS. Tris, 5 mM EDTA, and 0.02% NaN3. ImThe radioiodinated cells were lysed in PBS mune complexes were eluted from the S. containing 0.5% sodium deoxycholate, aweus by heating at 100” for 4 min in a 0.1% sodium dodecyl sulfate (SDS), and sample buffer containing 0.01 M Tris-hy1% Triton X-100, or lysing buffer containdrochloride (pH 6.8), 1% SDS, 1% fl-mering 1% NP-40, 120 mM NaCl, and 50 mM captoethanol, 12.5% glycerol, 0.5% Triton Tris (pH 8.0). The lysates were clarified by X-100, 0.25% deoxycholate, and 0.01% centrifugation at 2000 g for 10 min, folbromphenol blue. lowed by 100,000 g for 1 hr. Samples were SDS-polyacrylamide gel electrophoresis stored at -7O’C. (SDS-PAGE). Immunoprecipitates were Immune precipitation Samples of the analysed by sodium dodecyl sulfate-polycell lysates were incubated for 1 hr or acrylamide gel electrophoresis according overnight at 4°C with various antisera (3to Laemmli (1970). Polyacrylamide sepa30 ~1 of undiluted serum per 20-200 ~1 of ration gels (10 or 12.5%) were made in 1.5 the lysate). This mixture was incubated M Tris-hydrochloride (pH 8.8) containing
8
2
2
2
4
2
ND
2 ND
rr
rr
9s
88
88
9s
88
85
9s
C4W
FRG
NZB/San
A/J
AKR/J
B6
BG-G,x+
123
12!+GIx-
2
2
4
2
2
2
+
-
-
-
-
-
+ -
+ + +
-
-
+
+
+
+
+ -
+ -
+
+
+
+
-
2 +
-
8
-
-
gp70
anti-
antiFLV
-
specific” antisera”
“groupspecific” antisera*
3
No. of mice
“Fv-4 type-
+
+
t +
+ +
+ +
-
antiRLV
+
+
+ -
+
‘%x-m70
Tung’s (Tung
-
-
+ -
-
ec+-gp70
classification et al, 1978)
+ -
+
ND ND
o-gp70
Note. Surface of thymocytes from 4- to 12-month-old mice were labeled with ‘=I The cell lysates were immune precipitated with the five types of antisera indicated, and the precipitates were analyzed by SDS-PAGE. Positive (+) or negative (-) reaction indicates presence or absence of radioactive precipitates at Af, 70K to SOK on the gel. Reactivities of these five typing sera to MuLV viral antigen were described under Materials and Methods. D “Fv-4 type-specific” antisera means some lots of anti-Fv-4w’” and anti-C4W sera showing reactivity to C4W thymocyte gp70-related antigen but not to AKR thymocyte gp70-related antigen. (See Materials and Methods). *“Group-specific” antisera means other lots of anti-Fv-C, anti-G, and anti-C4W sera showing reactivity to both C4W and AKR thymocyte gp70-related antigens.
5
no. of tested
89
Total mice
BALB/c
Fv-4
genotype
Strain
2
Reactions of antisera with lysates of surface labeled thymocytes from various mouse strains
TABLE
g
F
Fi
Fv-h GENE AND CELL MEMBRANE @i'O
0.1% SDS. Polyacrylamide stacking gels (4.5%) were made in 0.5 M Tris-hydrochloride (pH 6.8) containing 0.1% SDS. Electrophoresis was performed at 40 V constant voltage for about 16 hr. After electrophoresis, gels were stained with Coomassie brilliant blue in solution of 10% acetic acid and 40% methanol, and then destained in the same solution without the dye. Gels were dried and exposed to X-ray film (Kodak XR-2 or Fuji XR) at -70°C. Lectin binding assay for cell surface antigen. Con A-Sepharose 4B (Pharmacia Fine Chemicals) was suspended in a stock solution (0.1 M acetate buffer, pH 6.0, containing 1 M NaCl, 1 mM MnClz, 1 mM MgC12, and 0.01% merthiolate) and washed with a reaction buffer (1% NP-40,120 mM NaCl, 50 mM Tris, 1 mM CaClz, and 1 mM MnCl,, pH 8.0) by centrifugation. Eighty microliters of the lysate of iodinated C4W thymocytes was suspended in the lysing buffer and mixed with an equal volume of packed Con A-Sepharose 4B beads (about 0.8 mg Con A). The mixture was incubated on ice for 45 min, and the beads were washed four times with the reaction buffer. The supernatants were collected and the washed Con A-Sepharose 4B beads divided into two aliquots. They were further washed with either the reaction buffer or the same buffer containing 0.5 Ma-methylD(+)-glucoside. These elutions (supernatants) were immune-precipitated and analyzed on SDS-PAGE. RESULTS
Cell surface antigens of BALB/c and a partially umgenic mice BALB/c-Fv-4-w’ (UW). Thymocytes from BALB/c (Fv-4”) and C4W (Fv-4”) mice were labeled by the lactoperoxidase-catalyzed iodination with ‘=INa, and the radiolabeled cell extracts were then analyzed by immune precipitation and SDS-PAGE. Figure 1 shows the immune precipitates from BALB/c and C4W lysates. Many radioactive components common to both mice were shown. However, with anti-Fv-4w’” (lane l), anti-G (lane 2), anti-FLV (lane 3), and anti-RLV (lane 4) sera, at least two unique proteins were precipitated from the
131
lysate of C4W mice. The apparent molecular weights of these precipitates were 70,000-85,000 and 15,000. All C4W mice tested showed the same major precipitate of 70-80K protein but the 15K protein was often very faint. A few BALB/c mice expressed a small quantity of a 73K dalton precipitate reactive with the anti-RLV and the anti-gp70 sera (Table 2). Anti-MuLV, serum gave almost an identical pattern for the two strains (lane 5). The fact that the 70-85K protein on the C4W thymocytes was precipitated with the anti-RLV serum (Fig. 1, lane 4) suggests that the protein shares antigenic determinants with some MuLV structural components. To test this, several types of goat antisera directed against purified proteins of MuLV were examined in the immuneprecipitation assay. As shown in Fig. 2, antiserum to gp70 (lane 2) precipitated a 70-85K protein as did the anti-RLV (lane 1 and 9). In contrast, antisera to gag gene products ~30, ~15, ~12, and ~10 did not precipitate the 70-80K protein. This indicated that the 70-85K protein may be antigenically related to the env gene product of MuLV. We noticed that mouse antisera, antiFv-4wrs, anti-G, and anti-FLV, precipitated a protein with a molecular weight of approximately 80K, while goat anti-gp70 and rabbit anti-RLV gave a precipitate ranging broadly from 70K to 85K (Figs. 1 and 2). For further analysis of these precipitates, we carried out the following absorption tests. (A) The lysate of the iodinated C4W thymocytes was first reacted with anti-gp70 antiserum, and the immune complexes were collected by Staphylococcus aureus. With this pretreatment, the 70-85K molecule was precipitated as described above (lane 1 of Fig. 3A). The supernatant was divided into two parts, and then mixed either with anti-Fv-4w’” serum or with anti-FLV serum. Neither of these antisera precipitated proteins in the 70-85K region on the gel (lanes 2 and 3). (B) Conversely, when the 80K protein was first precipitated by anti-Fv-4w” (lane 1 of Fig. 3B), the supernatant contained only a smaller protein (73K) which was precipitated with the anti-gp70 (lane 2) but not
132
IKEDA
AND
ODAKA
BA LB/c 1234512345
25
FIG. 1. Autoradiograph of immune precipitates of iodinated ceil surface proteins. Lysates of rwIlabeled BALB/c thymocytes and C4W thymocytes were used in immune-precipitation assays with: (1) anti-Fv-4wn serum (Lot 1121); (2) anti-G serum (Lot 1210); (3) anti-FLV serum; (4) anti-RLV serum; and (5) anti-MuLV.. The immune precipitates were anaiysed by eiectrophoresis on 12.5% polyacryiamide gel. The arrow indicates the location of poorly precipitated 15K antigens of C4W thymocytes. The positions of molecular weight markers were indicated at the right (X103 Af,).
with anti-FLV serum (lane 3). (C) Similarly, the lysate preabsorbed with antiFLV serum (lane 1 of Fig. 3C) contained only a 73K protein precipitable with antigp70 antiserum (lane 2) but the 80K band was no longer demonstrable by anti-Fv4w’* serum (lane 3). Identical results were obtained from a similar absorption test with a different combination of antisera, anti-C4W, anti-G, and anti-RLV (data not shown). These results revealed that at least two gp70-related molecules were expressed on the cell membrane of C4W thymocytes, and that they are distinct from each other in molecular weight and antigenicity. The 80K protein carried antigenie determinants detected by all five antisera, anti-Fv-4w’“, anti-C4W, anti-G, anti-FLV, anti-gp70, and anti-RLV, and the other ‘73K protein carried antigenic determinants detected by only anti-gp’70 and anti-RLV sera.
The presence of ?‘3K and SOK molecules on the surface of thymocytes are closely linked to the Fv-4’ gene. The C4W mouse is partially congenic for Fv-4’ and carries a region of chromosome 12 from the wild mouse which includes the Fv-4 and the Pre1 loci (Odaka et ah, 1981). In order to establish the genetic association of the cell surface antigens with the Fv-.&controlled resistance to FLV, we carried out a genetic study. A mouse heterozygous for Fv-4 was backcrossed to BALB/c and 7 progeny were tested by thymectomy for the presence or absence of these surface antigens. These hybrids mice were expected to be either of Fv-4” or Fv-4” genotype. Ten days after thymectomy the mice were inoculated with FLV, and sacrificed 10 days later. Their spleens were weighed and homogenized to be tested for LLV. As shown in Fig. 4, three mice (Nos. 1, 5, and 6) expressed the 80K and the 73K antigens. Their precipitation
Fv-l,
GENE
AND
CELL
MEMBRANE
133
gp70
60
25
'12.5 FIG. 2. Autoradiograph of immune precipitates of C4W iodinated cell surface protein. Lysate of ‘“SI-labeled C4W thymocytes was immune precipitated with anti-RLV serum (lanes 1 and 9). antigp70 serum (lane Z), anti-p30 serum (lane 3), anti-p15 serum (lane 4), anti-p12 serum (lane 5), anti~10 serum (lane 6), anti-Fv-4w” serum (lot 85) (lane 7), and anti-G serum (Lot 1210) (lane 8). The precipitates were analyzed by electrophoresis on 12.5% polyacrylamide gel.
patterns were identical to those found in the C4W mice, and all three mice were resistant to FLV infection (~13 PFU/spleen) and FLV-induced splenomegaly induction (co.3 g). In contrast, four mice (Nos. 2, 3, 4, and 7) that were susceptible to FLV infection (>7 X lo5 PFU/spleen) and FLVinduced splenomegaly induction (>0.53 g) did not express anti-C4W serum-precipitable antigen, but expressed a low and variable level of 73K antigen detectable with the anti-gp70 serum. In particular, mouse No. 7 expressed more 73K antigen than any BALB/c mice tested today (data not shown). Thus, the expression of the 80K molecule on the thymocytes was clearly a dominant trait and controlled by a locus very closely linked to the Fv-4’ allele; expression of the 73K molecule was not associated with resistance. As a control, each lysate was immune precipitated with the anti-H@ serum. This antiserum precipitated predominantly two proteins with molecular weights of 32,000
and 29,000, which correspond to (Ychains and p chains encoded by I-A and I-E subregions within the H-Z complex (Cullen et al., 1976, Silver et aZ., 1976). Other experiments demonstrating that this antiserum also carries natural antibodies to MuLV gp70 (data not shown) may explain why this serum precipitated an 80K antigen in the three resistant mice. Strain diference of gp7’0-related antigens on thymocytes. Cell surface antigens re-
lated to the gp7Os of MuLV have been found on thymocytes from many inbred strains of mice. Tung et al. (1978; 1976) classified thymocyte gp7Os of various mouse strains into three groups (GIx-gp70, ec+-gp70, Ogp70) by genetic and biochemical studies (see the reference in the right half of Table 2). We compared the 80K and 73K antigens with classified membrane gp7Os by serological analysis. During a survey of various lots of antiFv-4wrS, anti-G, and anti-C4W sera, we found that individual pooled serum showed
134
IKEDA
1
AND
ODAKA
A
El
231
2
C 31
2
80 K73 K-
FIG. 3. Sequential immune precipitation of iodinated surface proteins from C4W thymocytes. (A) Lysate of iodinated C4W thymocytes was immune precipitated with anti-gp70 serum (lane 1). The immune complex was cleared by adding fixed S. aurew..s and centrifuging. The unprecipitated supernatant was divided into two parts. They were further immune precipitated either with antiFv-4~‘” serum (Lot 1121) (lane 2) or with anti-FLV serum (lane 3). (B) The lysate was immuneprecipitated with anti-Fv-4” serum (Lot 1121) (lane l), and then the unprecipitated supernatant was further immune-precipitated either with anti-gp’70 serum (lane 2) or with anti-FLV serum (lane 3). (C) The lysate of iodinated C4W thymocytes was immune precipitated with anti-FLV serum (lane l), and then the unprecipitated supernatant was further immune precipitated either with goat anti-gp70 serum (lane 2) or with BALB/c anti-Fv-4w” serum (Lot 1121) (lane 3). The precipitates were analyzed by electrophoresis on 10% polyacrylamide gel.
either of two different specificities, termed as “group specific” or “Fv-4 type specific”. The group-specific antisera precipitated gp70-related antigens on both AKR thymocytes and C4W thymocytes, while the Fv-4 type-specific antisera precipitated only the 80K antigen on the C4W thymocytes (see Materials and Methods). We used a panel of five antisera in the following serological comparison for membrane gp7Os: “Fv-4 type-specific,” “groupanti-FLV, anti-gp70, and antispecific,” RLV sera. The reactivities of these sera for MuLV viral antigens were briefly described under Materials and Methods. Table 2 represents the serological reactivities of thymocyte gp7Os from eight mouse strains. The mouse strains could be largely classified into four groups. The first
group (some of BALB/c) did not express such a molecule, the second (some BALB/ c, NZB, A/J, B6, and 129) expressed membrane gp70 recognized by only anti-gp70 and anti-RLV, the third (AKR) expressed membrane gp70 recognized by the last four antisera, and the fourth (C4W ,and FRG) expressed membrane gp7Os recognized by all five antisera. A membrane gp70 expressed on BALB/ c thymocytes showed the same size and serological reactivities as the 73K antigen on C4W thymocytes. However, this antigen could be detected in only two of five BALB/c mice (Table 2) and if detected was in levels less than C4W mice (data not shown). FRG mice are the sole laboratory strain carrying the Fv-4’ allele, and two mem-
LLV (PFUlspleen) 7.0x105
041 1.5x106
1.31
NO-3
9.5x105
0.53
NO.4
0.30
NO.5
0.1 5
NO.6
6.2~10"
2.10
NO-7
FIG. 4. Genetic linkage test of the cell surface antigens with resistance to FLV infection in a backcross generation. Seven progeny from a cross of BALB/ c female with a heterozygous male mouse (Fv-4~“) were thymectomized at about 4 weeks. The surface of thymocytes from individual progeny was labeled with i”I, and then the cell lysates were immune precipitated with anti-G4W serum (Lot 41ml) (lane l), with anti-gp70 serum (lane 2), and with anti-H@ serum (lane 3). The precipitates were analyzed by electrophoresis on 10% polyacrylamide gel. Ten days after thymectomy, the mice were injected intraperitoneally with FLV (1.9 X 10’ PFU and 1.5 X lo3 FFU). Ten days later they were killed, and the individual spleens were weighed and homogenized to be tested for LLV. In general, we scored animals as susceptible to FLV when their spleens weighed more than 0.4 g.
0.16
Spleen wt.(g)
30K
80K 73K
NO.1 NO-2 123123123123123123123
?s $ ’
136
IKEDA
AND
brane gp7Os were detected on FRG thymocytes by absorption tests similar to those in Fig. 3 (data not shown). They were indistinguishable in molecular weight and antigenicity from the 80K and 73K antigens of C4W mice. AKR mice expressed two size classes of membrane gp7Os which were demonstrated in absorption test (Fig. 5). The larger gp70 (77K) appeared to be serologically related, but not identical, to the 80K antigen of C4W mice since the 77K antigen was not recognized by “Fv-4 type-specific” antiserum (Table 2). The smaller gp70 (72K) was serologically indistinguishable from the 73K antigen of C4W thymocytes. It should be stressed that the 77K antigen of AKR mice has the same serological reactivity as ecotropic viral gp7Os in our same assay system. The %3K and 8OK antigens of C4 W thymocytes were glycoproteins. To determine whether the 73K and 80K antigens of C4W thymocytes, though antigenically related to MuLV gp70, are glycosylated proteins, we analyzed the lectin-binding capacity of the two antigens. Since Con A specifically binds cy-D-manopyranoside or a-D-glucopyranoside, Con A-Sepharose 4B was mixed with the lysate of iodinated C4W thymocytes and washed with lysing buffer. The 73K and 80K antigens were not detected in the supernatant, but successfully eluted from the Con A-Sepharose 4B with a-methyl-D(+)-glucoside-containing lysing buffer (data not shown). This suggests that the Fv-.&‘-associated 73K and 80K molecules were glycoproteins. DISCUSSION
This comparative study between BALB/ c and its Fv-4 congenic C4W suggests that Fv-$-mediated resistance is associated with the expression of MuLV gp70-related cell surface antigens. Several distinct classes of antigens were identified on thymocytes from C4W mice with molecular weights of approximately 80K, 73K, and 15K. The 80K and 73K proteins were found to be glycosylated and serologically related to MuLV gp70. Genetic experiments demonstrated that the 80K antigen seg-
ODAKA
-94
-68 -45
FIG. 5. Sequential immune precipitation analysis of iodinated surface proteins from AKR/J thymocytes. (A) Three identical lysates of ‘%I-labeled AKR/ J thymocytes were immune precipitated either with anti-Fv-4w’” serum (Lot 85) (A lane l), with anti-G serum (Lot 1210), (B lane l), with anti-RLV serum (C lane 1). The immune complexes were cleared by adding fixed S. aureus and centrifuging. Each of the unprecipitated supernatants was further immuneprecipitated with anti-RLV serum (A lane 2), with anti-RLV serum (B lane 2), or with anti-G serum (lot 1210) (C lane 1). The immune precipitates were analyzed by electrophoresis on 10% polyacrylamide gels.
regated with Fv-Q-controlled resistance to FLV infection. A quantitative difference in the expression of the 73K antigen was detected between this congenic pair, but this difference was not associated with resistance in the backcross mice (Fig. 4). Thus, the Fv-4 gene may represent structural information for a unique cell surface gp70 or a regulatory locus which governs expression of this gp70. The dominant resistant allele at the Fv4 locus confers resistance to infection of FLV and ecotropic MuLVs (Kai et al, 1976; Odaka et al, 1981), and also appears to specify the expression of membrane gp70.
Fv-l
GENE
AND
CELL
The Fv-4 gene is thought to influence certain viral replication steps specific for ecotropic MuLV (Kai et a& 1976; Yoshikura et ul, 19’79; Odaka et aL, 1981). In the first step of virus infection, ecotropic and amphotropic MuLVs bind individually the specific receptors of mouse cells, respectively controlled by the Real gene (chromosome 5) (Oie et aZ., 1978; Ruddle et aL, 1978; Marshall and Rapp, 1979) and RumI gene (chromosome 8) (Gazdar et al, 1977), although these specific receptors have yet to be defined. The receptor binding component of MuLV is the major envelope glycoprotein gp70 (Gazdar et uk, 1977; Oie et al., 1978; Ruddle et al., 1978; Marshall and Rapp, 1979). Thus, the most simple hypothesis we can envision for Fv-h-mediated resistance is a viral interference model: in Fv-&resistant mice the specific receptor on cell membranes to ecotropic MuLVs might be already bound by the Fv4’-controlled gp7Os, and therefore the binding of exogenous ecotropic MuLVs is inhibited. This model also could explain the Fv-4’-mediated suppression of endogenous ecotropic MuLV expression in hybrids of AKRxFRG (Odaka et CL& 1980), because, if the endogenous AKR proviruses are activated in the hybrid mice, their secondary spread to surrounding cells is obstructed. This proposed mechanism for Fv-Q-mediated resistance is not unique; other systems have been described in which resistance can be correlated with the expression of viral gp7Os. The avian genes h-E, ev3, and ev6 control both cellular expression of the env gene of the endogenous subgroup E provirus and host resistance to exogenous subgroup E virus infection (Robinson and Lamoreux, 1976; Ando and Toyoshima, 1976; Robinson et al, 1981). The resistance of several mouse strains to Friend MuLV-induced erythroproliferative disease was correlated with the endogenous expression of an MCF/xenotropic virus gp70-related proteins on the surface of cells of resistant mice (Ruscetti et al, 1981; Bassin et uL, 1982). Using the Tung classification of the cell membrane gp7Os (Tung et aL, 1978; see Table 2), our data suggest that the Fv-4’as-
MEMBRANE
gp’70
137
sociated 73K protein is antigenically related to GIx-gp70 of 129 mice or 0-gp7O of B6 mice, while the 80K antigen is a new type of membrane gp70, but probably closely related to ec+-gp70 of AKR mice (Table 2). Several other gp70-related cell surface antigens (e.g., GIx (Stockert et uL, 1971), G~RADAI) (Obata et cd, 19W, G(AKSL~) (Stockert et al, 1979) GcEmn) (Obata et al, 1981), and XenCSA (Morse et &, 1979) have been identified on mouse lymphocytes by cytotoxic assay or membrane immunofluorescence using specific antisera. Though we have not directly compared these antigens with the Fv-$‘-associated 80K antigen, the strain distributions of these other antigens are clearly distinct from that of the Fv-J-linked antigen (Table 2). ACKNOWLEDGMENTS We thank K. Kai, H. Yoshikura, and the Division of Cancer Cause and Prevention, NCI, for providing us with antisera; S. Suzuki, K. Suzuki, and T. Noguchi for providing us with inbred strains of mice; M. Yamashita, K. Asahi, and Y. Kagami for technical assistance; H. Sato, Y. Sanai, and F. Taguchi for reading this manuscript and helpful discussion; and N. Munezawa for preparation of the manuscript. We are grateful to people at the National Institutes of Health, especially C. A. Kozak, W. Y. Langdon, and H. C. Morse, III for their correction of English of this manuscript and S. Grove for the preparation of the manuscript. This research was supported in part by a grant from Japanese Ministry of Education and by a grant from the Mitsubishi Foundation.
REFERENCES ANDO, T., and TOYSHIMA, K. (1976). Genetic control of chick helper factor in cells which lack natural group-specific antigen of avian leukosis. VirdoQy 73.521-52’7. BASSIN, R. H., RUSCETTI, S., ALI, I., HAAPALA, D. K., and REIN, A. (1982). Normal DBA/Z mouse cells synthesize a glycoprotein which interferes with MCF virus infection. Virology 123, 139-151. BASSIN, R. H., TUNE, N., and FISCHINGER, P. J. (1971). Rapid cell culture assay technique for murine leukaemia viruses. Nature &n&m) 229,5644X6. CULLEN, S. E., FREED, J. H., and NATHENSON, S. G. (1976). Structural and serological properties of murine Ia alloantigens. Transplant. Rev. 29,236-270. GARDNER, M. B., RASHEED, S., PAL, B. K., Ems, J. D., and O’BRIEN, S. J. (1980). Ahn-I, a dominant murine leukemia virus restriction gene, is poly-
138
IKEDA
morphic in leukemia-prone Acad
Sci
AND ODAKA
wild mice. Proc. Nat.
USA 77,531-535.
GAZDAR, A. F., OIE, H., LALLEY, P., Moss, W. W., and MINNA, J. D. (1977). Identification of mouse chromosomes required for murine leukemia virus replication. CeU 11, 949-956. IKEDA, H., SATO, H., and ODAKA, T. (1981). Mapping of the Fv-.4 mouse gene controlling resistance to murine leukemia viruses. Ink J. Cancer 28,2X7-240. KAI, K., IKEDA, H., YUASA, Y., SUZUKI, S., and ODAKA, T. (1976). Mouse strain resistant to N-, B-, and NBtropic murine leukemia viruses. J. Vird 20, 436440.
LAEMMLE, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-687. MARSHALL, T. H., and RAPP, U. R. (1979). Genes controlling receptors for ecotropic and xenotropic type C virus in Mus ceruicolor and Mus musculus. J. Virol 29,501-506. MORSE III, H. C., CHUSED, T. M., BOEHM-TRUITT, M., MATHIESON, B. J., SHARROW, S. O., and HARTLEY, J. W. (1979). XenCSA: Cell surface antigens related to the major glycoproteins (gp70) of xenotropic murine leukemia viruses. J. Immurwl 122.443-454. OBATA, Y., STOCKERT, E., O’DONNEU, P. V., OKUBO, S., SNYDER,H. W. JR., and OLD, L. J. (1978). GcmAn: A new cell surface antigen of mouse leukemia defined by naturally occurring antibody and its relationship to murine leukemia virus. J. Exp. MecL 147.1089-1105. OBATA, Y., STOCKERT, E., DELEO, A. B., O’DONNELL, P. V., SNYDER, H. W. JR., and OLD, L. J. (1981). G (aRLn):A cell surface antigen of the mouse related to xenotropic MuLV defined by naturally occurring antibody and monoclonal antibody. J. Exp. Med 154.659-675. ODAKA, T., and MATSUKURA, M. (1969). Inheritance of susceptibility to Friend mouse leukemia virus. VI. Reciprocal alteration of innate resistance or susceptibility by bone marrow transplantation between congenic strains. J. Viral. 4, 337-343. ODAKA, T., IKEDA, H., MORIWAKI, K., MATSUZAWA, A., MIZUNO, M., and KONDO, K. (1978). Genetic resistance in Japanese wild mice (Mus musculus molossinus) to an NB-tropic Friend murine leukemia virus. J. Nat. Cancer Inst. 61,1301-1306. ODAKA, T., IKEDA, H., and AKATSUKA, T. (1980). Restricted expression of endogenous N-tropic XCpositive leukemia virus in hybrids between G and AKR mice: An effect of the Fv-l’ gene. Int. J. Can~~-25.757-762.
ODAKA, T., IKEDA, H., YOSHIKURA, H., MORIWAKI, K., and SUZUKI, S. (1981). Fv-6: Gene controlling resistance to NB-tropic Friend murine leukemia virus. Distribution in wild mice, introduction into genetic background of BALB/e mice, and mapping of chromosomes. J. Nat. Cancer fnst 67,1123-1127.
OIE, H. K., GAZDAR, A. F., LALLEY, P. A., RUSSELL, E. K., and MINNA, J. D. (1978). Mouse chromosome 5 codes for ecotropic murine leukaemia virus eellsurface receptor. Nature (London) 274, 60-62. OLD, L. J., and STOCKERT, E. (1977). Immunogenetics of cell surface antigens of mouse leukemia. Annzl Rev. Genet. 11, 127-160. ROBINSON, H. L., and LAMOREUX, W. F. (1976). Expression of endogenous ALV antigens and susceptibility to subgroup E ALV in three strains of chickens (Endogenous avian C-type virus). viralogy 69,50-62. ROBINSON, H. L., ASTRIN, S. M., SENIOR, A. M., and SALAZAR, F. H. (1981). Host susceptibility to endogenous virus: Defective, glycoprotein-expressing proviruses interfere to infections. .J. viral. 40,745751. ROWE, W. P., PUGH, W. E., and HARTLEY, J. W. (1970). Plaque assay techniques for murine leukemia viruses. virology 42, 1136-1139. RUDDLE, N. H., CONTA, B. S., LEINWAND, L., KOZAK, C., RUDDLE, F., BESMER, P., and BALTIMORE, D. (1978). Assignment of the receptor for ecotropic murine leukemia virus to mouse chromosome 5. J. Exp. Med 148,451-465. RUSCE~, S., DAVIS, L., FE&D, J., and OLIFF, A. (1981). Friend murine leukemia virus-induced leukemia is associated with the formation of mink cell focusinducing viruses and is blocked in mice expressing endogenous mink cell focus-inducing xenotropic viral envelope genes. J. Exp. Med 154, 907-920. SILVER, J., CECKA, J. M., MCMILLAN, M., and HOOD, L. (1976). Chemical characterization of products of the H-2 complex. Cold Spring Harbor Symp. Quad. Biol. XLI, 369-377. STOCKERT,B., OLD, L. J., and BOYSE, E. A. (1971). The GIx system. A cell surface allo-antigen associated with murine leukemia virus: Implications regarding chromosomal integration of the viral genome. J. Exp. Med 133,1334-1335. STOCKERT,E., DELEO, A. B., O’DONNELL, P. V., OBATA, Y., and OLD, L. J. (1979). GCAKSUj:A new cell surface antigen of the mouse related to the dualtropic mink cell focus-inducing class of murine leukemia virus detected by naturally occurring antibody. J. Esp. Med.
149,200-215.
SUZUKI, S. (1975). FV-4: A new gene affecting the splenomegaly induction by Friend leukemia virus. Japan. J. Exp. Med 45.473-478. SUZUKI, S., and MATSUBARA, S. (1975). Isolation of Friend leukemia virus resistant line from noninbred mouse colony. Japan. J. Exp. Med 45,467471. TUNG, J. S., SHEN, F. W., FLEISSNER, E., and BOYSE, E. A. (1976). X-gp70: A third molecular species of the envelope protein gp70 of murine leukemia virus, expressed on mouse lymphoid cells. J. Exp. Med
143,969-974.
Fv-4 GENE
AND CELL
TUNG, J. S., O’DONNELL, P. V., FLEISSNER, E., and BOYSE, E. A. (1978). Relationships of gp70 of MuLV envelopes to gp’70 components of mouse lymphocyte plasma membranes. J. Exp. Mea! 147, 12801284. WRIGHT, B. S., O’BRIEN, P. A., SHIBLEY, G. P., MAYYASI, S. A., and LASFARGUES, J. C. (1967). Infection of an established mouse bone marrow cell line (JLS-V9) with Rauscher and Moloney murine leukemia viruses. Cancer Res. 27,1672-1677. YOSHIDA, M., and YOSHIKURA, H. (1981). A glycoprotein specified by spleen focus-forming virus in three
MEMBRANE
gp70
139
cell lines varies in molecular size and peptide composition, but retains both xenotropic and ecotropic MuLV antigenicity. J. Gen ViroL 52, 183-188. YOSHIKURA, H., NAITO, H., and MORIWAKI, K. (19’79). Unstable resistance of G mouse fibroblasts to ecotropic murine leukemia virus infection. J. ViroL 29, 1078-1086. YOSHIKURA, H., and ODAKA, T. (1982). Surface antigen expressed in hematopoietic cells derived from Fv-4’ mouse strains. J. Nut. Cancer Inst. 68, 10051009.
128, 127-139
(1983)
Cellular Expression of Murine Leukemia Virus gp70-Related Antigen Thymocytes of Uninfected Mice Correlates with Fv-4 Gene-Controlled Resistance to Friend Leukemia Virus Infection HIDETOSHI Department
IKEDA’
of Genetics, Institute 4-6-l Shirokanedai, Received
August
AND of Medical Minabku,
TAKESHI
ODAKA of
Science, University Tokyo 108, Japan
25, 1982; accepted
March
on
Tokyo,
29, 1983
Alleles at the Fv-4 locus have been shown to determine susceptibility (Fv-P) and resistance (Fv-4’) to infection with ecotropic murine leukemia viruses (MuLV). The resistance is dominant in heterozygous mice. BALB/c mice are Fv-4” and a semicongenic line BALB/c-Fv-hw’, homozygous for Fv-4’ allele and called C4W, is being developed. Extracts of ‘l-labeled thymocytes from BALB/c and C4W mice were precipitated by a series of antisera against MuLV and the precipitates examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Thymocytes from C4W mice expressed 80K and 73K glycoproteins related to the gp70 of MuLV, while only the 73K glycoprotein was occasionally detectable on BALB/c thymocytes. Genetic studies showed that a locus controlling expression of the 80K protein was dominant and associated with the resistant allele at Fv-4. This membrane gp70 may be important in the Fv-h-mediated resistance to exogenous virus infection. INTRODUCTION
eluding FLV and the expression of endogenous ecotropic MuLV are almost completely restricted (Kai et aZ., 1976; Odaka et al., 1980; Gardner et al., 1980). Recently we established a partially congenie mouse line BALB/c-Fv-4~’ (C4W) which carries the Fv-4’ allele from a wild mouse on the BALB/c background (Odada et ah, 1981). During the course of establishing the congenic line, we immunized BALB/c mice repeatedly with lymphoid cells from progeny mice derived from crosses between BALB/c and BALB/c-FV..$w~‘, in an attempt to identify the gene product specified by the Fv-4’ allele. Some of the pooled antisera were found to recognize a cell surface alloantigen associated with the Fv-4’ allele (Yoshikura and Odaka, 1982). In this study we describe the characterization of this cell surface antigen. The results indicate that at least two MuLV gp70-related molecules are expressed on the plasma membranes of thymocytes of C4W mice, one of which appears unique to previously described membrane gp7Os (Old and Stockert, 1977; Tung et al., 1976). The
The Fv-4 locus controls susceptibility to Friend leukemia virus (FLV) infection and also influences sensitivity to a variety of ecotropic murine leukemia viruses (MuLVs) (Suzuki, 19’75; Suzuki and Matsubara, 1975; Kai et al., 1976; Yoshikura et ab, 19’79; Odaka et ah, 1981). This locus is located on chromosome 12 (Ikeda et al., 1981) and has two alleles, Fv-4” (susceptibility) and Fv-4’ (resistance). The dominant resistance allele was found in one strain of laboratory mice (Suzuki, 1975) and in many wild Asian mice (Odaka et al., 1978), and is presumably identical to the Akvr-1 locus described by Gardner et al. (1980) in wild California mice. In Fv-4’ mice and Akvr-1’ mice, the replication of exogenously infected ecotropic MuLVs in’ Present Address: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 20205. Author to whom requests for reprints should be addressed. 127
0042-6822183 Copyright All rights
$3.00
CC 1983 by Academic Press, Inc. of reproduction in any form reserved
128
IKEDA
AND
identification of such a cell surface gp70 molecule provides a possible explanation for Fv-&associated resistance. MATERIALS
AND
METHODS
Mice. BALB/c, AKR/J,and C57BL/6(B6) mice were bred in our laboratory. FRG mice, previously called G mice (Suzuki, 1975; Suzuki and Matsubara, 1975), and A/ J mice were provided by Dr. S. Suzuki, Research Laboratories, Chugai Pharmaceutical Company, Ltd., Tokyo, Japan. NZB/ San mice were obtained from the Laboratory of Experimental Animals of our Institute. 129/Sv-SZCp(129) mice were a kind gift from Dr. T. Noguchi, National Institute of Genetics, Mishima, Japan. The partially congenic mouse line BALB/c-Fv-QW’ (C4W) was established by introducing the Fv-4’ gene from a wild mouse onto the BALB/c (Odaka et al, 1981). Mice used here were at the sixth backcross generation. C57BL/6-Fv” is a congenic strain carrying the Fv-2” allele of DDD mice on the C57BL/ 6 background (Odaka and Matsukura, 1969). BALB.D is a congenic strain carrying the H-Z complex of DDD mice on the BALB/c background (Ikeda, unpublished data). All the mice used for testing thymocyte antigens were 3 to 12 weeks old and of both sexes. Antisera. The antisera used in this work are listed in Table 1. In this report, we describe two different types of specificity in the three Fv-4 alloantisera (anti-Fv4wTs,anti-G, and anti-C4W sera). One precipitated gp70-related cell surface antigens on both C4W and AKR thymocytes, while the other defined a gp70-related thymocyte antigen which is unique to Fv-4’ mice. We tentatively term these “groupspecific antisera” and “Fv-4 type-specific antisera,” respectively (see Table 2). The specificity of anti-FLV serum was indistinguishable from that of the group-specific antiserum. By each recipient-donor combination (BALB/c anti-Fv-4’“, (BALB/ c X DDD)Fi anti-G, and BALB/c antiC4W), both group-specific and Fv-4 typespecific antisera were obtained. However, to avoid confusion, we used a limited number of antisera lots throughout this work;
ODAKA
anti-Fv-4w’” (Lots 85 and 1121) and antiC4W (Lots 41ml and 41m4) as Fv-4 typespecific antisera, and anti-G (Lot 1210) as group-specific antiserum. All of these antisera were used without special absorption. We used a panel of five antisera in experiments of Table 2. The reactivities of these sera for MuLV antigens were tested (Ikeda and Odaka, manuscript in preparation). The “Fv-4 type-specific” antisera precipitated none of viral proteins tested. The “group-specific” and anti-FLV sera precipitated gp7Os from all ecotropic and some dualtropic MuLV strains but no xenotropic MuLVs. The anti-gp70 and antiRLV sera precipitated gp7Os from all ecotropic, xenotropic, and dualtropic MuLV strains tested. P15(E)s from all ecotropic, dualtropic, and some xenotropic MuLV strains were precipitated by “group-specific,” anti-FLV, and anti-RLV sera, and weekly by anti-gp70. Most gag proteins of all MuLV strains tested were precipitated only by anti-RLV. Virus and virus assay. NB-tropic Friend leukemia virus (FLV) that had been serially passaged in BALB/c mice was prepared from the spleen homogenate of virus-infected BALB/c mice. The lymphatic leukemia virus (LLV) titer was measured on C-182 cells (Bassin et al, 1971), using the XC-plaque assay (Rowe et aL, 1970). The titer was expressed as PFU/spleen. Linkage analysis of cell surface antigens on the thywwcytes with the Fv-4 genotype. A mouse of C4W line, which was heterozygous for Fv-4 and at the ninth backcross generation, was backcrossed to a BALB/c mouse. The hybrid mice were thymectomized at about 4 weeks of age. Thymocytes were used for the surface iodination. Ten days after thymectomy mice were inoculated intraperitoneally with 0.2 ml of NBtropic FLV (1.5 X lo3 FFU and 1.9 X lo5 PFU). Individual spleens were weighed 10 days later and homogenates titered for LLV. Cell surface iodinaticm. Thymocytes suspended at a concentration of l-2.5 X 10’ per ml in 0.1 or 0.2 ml phosphate-buffered saline (PBS) were iodinated by incubation with 50 fig of lactoperoxidase (Boehringer
h-6
GENE
AND
CELL TABLE
MEMBRANE
gp70
129
1
ANTISERA USED Abbreviation Anti-Fv-4w”
Anti-G Anti-C4W Anti-H-2 Anti-FLV Anti-RLV
Immunization
and descriptions
BALB/c mice were immunized with normal spleen, thymus, and lymph node cells from Fv-4” and Fv-4” mice which were born during the course to establish the congeneic C4W mice, Some of the pooled sera contain cytotoxic antibody to a cell surface antigen linked to the Fv-4’ allele (H. Yoshikura and T. Odaka, 1982). (BALB/c X DDD)F, mice were immunized with spleen cells from G (FRG) mice with Fv4” genotype. BALB/c mice were immunized with spleen cells and thymocytes from C4W mice. BALB.D mice were immunized with spleen cells from BALB/c mice. This alloantiserum contained antibodies to H-zd antigens as revealed by hemagglutination test and a low titer of antibody against gp70 of MuLV (data not shown). C57BL/6-Fv” mice were inoculated with N-tropic FLV, developed, and then recovered from splenomegaly. These mice were bled and the sera pooled. This antiserum precipitates gp70 and p15(E) of FLV (Dr. K. Kai, personal communication). A rabbit was immunized with purified RLV produced by JLS-V9 cells (Wright et al, 1967). This was a kind gift from Dr. K. Kai.
Anti-MuLV,
A rabbit was immunized with SiRC cells,a rabbit cell line, infected with xenotropic MuLV (Yoshida and Yoshikura, 1981), as described by Morse et al (1979). This was a kind gift from Dr. H. Yoshikura.
Anti-gp70 Anti-p30 Anti-p15 Anti-p12 Anti-p10
Goats were immunized with gp70, ~30, ~15, ~12, or p10 purified from RLV. These were kind gifts of The Division of Cancer Cause and Prevention, NCI.
Mannhaim) and 0.5 mCi of Na’%I (New for formalin-fixed Staphylococcus aweus England Nuclear) at room temperature. at 4” for 60-120 min, and immune comThe reaction was initiated by the addition plexes were collected by centrifugation. of HaOa. Ten microliters of 0.03% of HaOa Precipitates were washed three times with was sequentially added at 2- to 5-min in- PBS containing 0.5% sodium deoxychotervals and the reaction terminated by late, 0.1% sodium dodecyl sulfate (SDS), washing the cells with RPM1 1640 medium and 1% Triton X-100, or NET buffer concontaining 5% precolostrum newborn calf taining 0.05% NP-40,150 mMNaC1,50 mM serum followed by two washes with PBS. Tris, 5 mM EDTA, and 0.02% NaN3. ImThe radioiodinated cells were lysed in PBS mune complexes were eluted from the S. containing 0.5% sodium deoxycholate, aweus by heating at 100” for 4 min in a 0.1% sodium dodecyl sulfate (SDS), and sample buffer containing 0.01 M Tris-hy1% Triton X-100, or lysing buffer containdrochloride (pH 6.8), 1% SDS, 1% fl-mering 1% NP-40, 120 mM NaCl, and 50 mM captoethanol, 12.5% glycerol, 0.5% Triton Tris (pH 8.0). The lysates were clarified by X-100, 0.25% deoxycholate, and 0.01% centrifugation at 2000 g for 10 min, folbromphenol blue. lowed by 100,000 g for 1 hr. Samples were SDS-polyacrylamide gel electrophoresis stored at -7O’C. (SDS-PAGE). Immunoprecipitates were Immune precipitation Samples of the analysed by sodium dodecyl sulfate-polycell lysates were incubated for 1 hr or acrylamide gel electrophoresis according overnight at 4°C with various antisera (3to Laemmli (1970). Polyacrylamide sepa30 ~1 of undiluted serum per 20-200 ~1 of ration gels (10 or 12.5%) were made in 1.5 the lysate). This mixture was incubated M Tris-hydrochloride (pH 8.8) containing
8
2
2
2
4
2
ND
2 ND
rr
rr
9s
88
88
9s
88
85
9s
C4W
FRG
NZB/San
A/J
AKR/J
B6
BG-G,x+
123
12!+GIx-
2
2
4
2
2
2
+
-
-
-
-
-
+ -
+ + +
-
-
+
+
+
+
+ -
+ -
+
+
+
+
-
2 +
-
8
-
-
gp70
anti-
antiFLV
-
specific” antisera”
“groupspecific” antisera*
3
No. of mice
“Fv-4 type-
+
+
t +
+ +
+ +
-
antiRLV
+
+
+ -
+
‘%x-m70
Tung’s (Tung
-
-
+ -
-
ec+-gp70
classification et al, 1978)
+ -
+
ND ND
o-gp70
Note. Surface of thymocytes from 4- to 12-month-old mice were labeled with ‘=I The cell lysates were immune precipitated with the five types of antisera indicated, and the precipitates were analyzed by SDS-PAGE. Positive (+) or negative (-) reaction indicates presence or absence of radioactive precipitates at Af, 70K to SOK on the gel. Reactivities of these five typing sera to MuLV viral antigen were described under Materials and Methods. D “Fv-4 type-specific” antisera means some lots of anti-Fv-4w’” and anti-C4W sera showing reactivity to C4W thymocyte gp70-related antigen but not to AKR thymocyte gp70-related antigen. (See Materials and Methods). *“Group-specific” antisera means other lots of anti-Fv-C, anti-G, and anti-C4W sera showing reactivity to both C4W and AKR thymocyte gp70-related antigens.
5
no. of tested
89
Total mice
BALB/c
Fv-4
genotype
Strain
2
Reactions of antisera with lysates of surface labeled thymocytes from various mouse strains
TABLE
g
F
Fi
Fv-h GENE AND CELL MEMBRANE @i'O
0.1% SDS. Polyacrylamide stacking gels (4.5%) were made in 0.5 M Tris-hydrochloride (pH 6.8) containing 0.1% SDS. Electrophoresis was performed at 40 V constant voltage for about 16 hr. After electrophoresis, gels were stained with Coomassie brilliant blue in solution of 10% acetic acid and 40% methanol, and then destained in the same solution without the dye. Gels were dried and exposed to X-ray film (Kodak XR-2 or Fuji XR) at -70°C. Lectin binding assay for cell surface antigen. Con A-Sepharose 4B (Pharmacia Fine Chemicals) was suspended in a stock solution (0.1 M acetate buffer, pH 6.0, containing 1 M NaCl, 1 mM MnClz, 1 mM MgC12, and 0.01% merthiolate) and washed with a reaction buffer (1% NP-40,120 mM NaCl, 50 mM Tris, 1 mM CaClz, and 1 mM MnCl,, pH 8.0) by centrifugation. Eighty microliters of the lysate of iodinated C4W thymocytes was suspended in the lysing buffer and mixed with an equal volume of packed Con A-Sepharose 4B beads (about 0.8 mg Con A). The mixture was incubated on ice for 45 min, and the beads were washed four times with the reaction buffer. The supernatants were collected and the washed Con A-Sepharose 4B beads divided into two aliquots. They were further washed with either the reaction buffer or the same buffer containing 0.5 Ma-methylD(+)-glucoside. These elutions (supernatants) were immune-precipitated and analyzed on SDS-PAGE. RESULTS
Cell surface antigens of BALB/c and a partially umgenic mice BALB/c-Fv-4-w’ (UW). Thymocytes from BALB/c (Fv-4”) and C4W (Fv-4”) mice were labeled by the lactoperoxidase-catalyzed iodination with ‘=INa, and the radiolabeled cell extracts were then analyzed by immune precipitation and SDS-PAGE. Figure 1 shows the immune precipitates from BALB/c and C4W lysates. Many radioactive components common to both mice were shown. However, with anti-Fv-4w’” (lane l), anti-G (lane 2), anti-FLV (lane 3), and anti-RLV (lane 4) sera, at least two unique proteins were precipitated from the
131
lysate of C4W mice. The apparent molecular weights of these precipitates were 70,000-85,000 and 15,000. All C4W mice tested showed the same major precipitate of 70-80K protein but the 15K protein was often very faint. A few BALB/c mice expressed a small quantity of a 73K dalton precipitate reactive with the anti-RLV and the anti-gp70 sera (Table 2). Anti-MuLV, serum gave almost an identical pattern for the two strains (lane 5). The fact that the 70-85K protein on the C4W thymocytes was precipitated with the anti-RLV serum (Fig. 1, lane 4) suggests that the protein shares antigenic determinants with some MuLV structural components. To test this, several types of goat antisera directed against purified proteins of MuLV were examined in the immuneprecipitation assay. As shown in Fig. 2, antiserum to gp70 (lane 2) precipitated a 70-85K protein as did the anti-RLV (lane 1 and 9). In contrast, antisera to gag gene products ~30, ~15, ~12, and ~10 did not precipitate the 70-80K protein. This indicated that the 70-85K protein may be antigenically related to the env gene product of MuLV. We noticed that mouse antisera, antiFv-4wrs, anti-G, and anti-FLV, precipitated a protein with a molecular weight of approximately 80K, while goat anti-gp70 and rabbit anti-RLV gave a precipitate ranging broadly from 70K to 85K (Figs. 1 and 2). For further analysis of these precipitates, we carried out the following absorption tests. (A) The lysate of the iodinated C4W thymocytes was first reacted with anti-gp70 antiserum, and the immune complexes were collected by Staphylococcus aureus. With this pretreatment, the 70-85K molecule was precipitated as described above (lane 1 of Fig. 3A). The supernatant was divided into two parts, and then mixed either with anti-Fv-4w’” serum or with anti-FLV serum. Neither of these antisera precipitated proteins in the 70-85K region on the gel (lanes 2 and 3). (B) Conversely, when the 80K protein was first precipitated by anti-Fv-4w” (lane 1 of Fig. 3B), the supernatant contained only a smaller protein (73K) which was precipitated with the anti-gp70 (lane 2) but not
132
IKEDA
AND
ODAKA
BA LB/c 1234512345
25
FIG. 1. Autoradiograph of immune precipitates of iodinated ceil surface proteins. Lysates of rwIlabeled BALB/c thymocytes and C4W thymocytes were used in immune-precipitation assays with: (1) anti-Fv-4wn serum (Lot 1121); (2) anti-G serum (Lot 1210); (3) anti-FLV serum; (4) anti-RLV serum; and (5) anti-MuLV.. The immune precipitates were anaiysed by eiectrophoresis on 12.5% polyacryiamide gel. The arrow indicates the location of poorly precipitated 15K antigens of C4W thymocytes. The positions of molecular weight markers were indicated at the right (X103 Af,).
with anti-FLV serum (lane 3). (C) Similarly, the lysate preabsorbed with antiFLV serum (lane 1 of Fig. 3C) contained only a 73K protein precipitable with antigp70 antiserum (lane 2) but the 80K band was no longer demonstrable by anti-Fv4w’* serum (lane 3). Identical results were obtained from a similar absorption test with a different combination of antisera, anti-C4W, anti-G, and anti-RLV (data not shown). These results revealed that at least two gp70-related molecules were expressed on the cell membrane of C4W thymocytes, and that they are distinct from each other in molecular weight and antigenicity. The 80K protein carried antigenie determinants detected by all five antisera, anti-Fv-4w’“, anti-C4W, anti-G, anti-FLV, anti-gp70, and anti-RLV, and the other ‘73K protein carried antigenic determinants detected by only anti-gp’70 and anti-RLV sera.
The presence of ?‘3K and SOK molecules on the surface of thymocytes are closely linked to the Fv-4’ gene. The C4W mouse is partially congenic for Fv-4’ and carries a region of chromosome 12 from the wild mouse which includes the Fv-4 and the Pre1 loci (Odaka et ah, 1981). In order to establish the genetic association of the cell surface antigens with the Fv-.&controlled resistance to FLV, we carried out a genetic study. A mouse heterozygous for Fv-4 was backcrossed to BALB/c and 7 progeny were tested by thymectomy for the presence or absence of these surface antigens. These hybrids mice were expected to be either of Fv-4” or Fv-4” genotype. Ten days after thymectomy the mice were inoculated with FLV, and sacrificed 10 days later. Their spleens were weighed and homogenized to be tested for LLV. As shown in Fig. 4, three mice (Nos. 1, 5, and 6) expressed the 80K and the 73K antigens. Their precipitation
Fv-l,
GENE
AND
CELL
MEMBRANE
133
gp70
60
25
'12.5 FIG. 2. Autoradiograph of immune precipitates of C4W iodinated cell surface protein. Lysate of ‘“SI-labeled C4W thymocytes was immune precipitated with anti-RLV serum (lanes 1 and 9). antigp70 serum (lane Z), anti-p30 serum (lane 3), anti-p15 serum (lane 4), anti-p12 serum (lane 5), anti~10 serum (lane 6), anti-Fv-4w” serum (lot 85) (lane 7), and anti-G serum (Lot 1210) (lane 8). The precipitates were analyzed by electrophoresis on 12.5% polyacrylamide gel.
patterns were identical to those found in the C4W mice, and all three mice were resistant to FLV infection (~13 PFU/spleen) and FLV-induced splenomegaly induction (co.3 g). In contrast, four mice (Nos. 2, 3, 4, and 7) that were susceptible to FLV infection (>7 X lo5 PFU/spleen) and FLVinduced splenomegaly induction (>0.53 g) did not express anti-C4W serum-precipitable antigen, but expressed a low and variable level of 73K antigen detectable with the anti-gp70 serum. In particular, mouse No. 7 expressed more 73K antigen than any BALB/c mice tested today (data not shown). Thus, the expression of the 80K molecule on the thymocytes was clearly a dominant trait and controlled by a locus very closely linked to the Fv-4’ allele; expression of the 73K molecule was not associated with resistance. As a control, each lysate was immune precipitated with the anti-H@ serum. This antiserum precipitated predominantly two proteins with molecular weights of 32,000
and 29,000, which correspond to (Ychains and p chains encoded by I-A and I-E subregions within the H-Z complex (Cullen et al., 1976, Silver et aZ., 1976). Other experiments demonstrating that this antiserum also carries natural antibodies to MuLV gp70 (data not shown) may explain why this serum precipitated an 80K antigen in the three resistant mice. Strain diference of gp7’0-related antigens on thymocytes. Cell surface antigens re-
lated to the gp7Os of MuLV have been found on thymocytes from many inbred strains of mice. Tung et al. (1978; 1976) classified thymocyte gp7Os of various mouse strains into three groups (GIx-gp70, ec+-gp70, Ogp70) by genetic and biochemical studies (see the reference in the right half of Table 2). We compared the 80K and 73K antigens with classified membrane gp7Os by serological analysis. During a survey of various lots of antiFv-4wrS, anti-G, and anti-C4W sera, we found that individual pooled serum showed
134
IKEDA
1
AND
ODAKA
A
El
231
2
C 31
2
80 K73 K-
FIG. 3. Sequential immune precipitation of iodinated surface proteins from C4W thymocytes. (A) Lysate of iodinated C4W thymocytes was immune precipitated with anti-gp70 serum (lane 1). The immune complex was cleared by adding fixed S. aurew..s and centrifuging. The unprecipitated supernatant was divided into two parts. They were further immune precipitated either with antiFv-4~‘” serum (Lot 1121) (lane 2) or with anti-FLV serum (lane 3). (B) The lysate was immuneprecipitated with anti-Fv-4” serum (Lot 1121) (lane l), and then the unprecipitated supernatant was further immune-precipitated either with anti-gp’70 serum (lane 2) or with anti-FLV serum (lane 3). (C) The lysate of iodinated C4W thymocytes was immune precipitated with anti-FLV serum (lane l), and then the unprecipitated supernatant was further immune precipitated either with goat anti-gp70 serum (lane 2) or with BALB/c anti-Fv-4w” serum (Lot 1121) (lane 3). The precipitates were analyzed by electrophoresis on 10% polyacrylamide gel.
either of two different specificities, termed as “group specific” or “Fv-4 type specific”. The group-specific antisera precipitated gp70-related antigens on both AKR thymocytes and C4W thymocytes, while the Fv-4 type-specific antisera precipitated only the 80K antigen on the C4W thymocytes (see Materials and Methods). We used a panel of five antisera in the following serological comparison for membrane gp7Os: “Fv-4 type-specific,” “groupanti-FLV, anti-gp70, and antispecific,” RLV sera. The reactivities of these sera for MuLV viral antigens were briefly described under Materials and Methods. Table 2 represents the serological reactivities of thymocyte gp7Os from eight mouse strains. The mouse strains could be largely classified into four groups. The first
group (some of BALB/c) did not express such a molecule, the second (some BALB/ c, NZB, A/J, B6, and 129) expressed membrane gp70 recognized by only anti-gp70 and anti-RLV, the third (AKR) expressed membrane gp70 recognized by the last four antisera, and the fourth (C4W ,and FRG) expressed membrane gp7Os recognized by all five antisera. A membrane gp70 expressed on BALB/ c thymocytes showed the same size and serological reactivities as the 73K antigen on C4W thymocytes. However, this antigen could be detected in only two of five BALB/c mice (Table 2) and if detected was in levels less than C4W mice (data not shown). FRG mice are the sole laboratory strain carrying the Fv-4’ allele, and two mem-
LLV (PFUlspleen) 7.0x105
041 1.5x106
1.31
NO-3
9.5x105
0.53
NO.4
0.30
NO.5
0.1 5
NO.6
6.2~10"
2.10
NO-7
FIG. 4. Genetic linkage test of the cell surface antigens with resistance to FLV infection in a backcross generation. Seven progeny from a cross of BALB/ c female with a heterozygous male mouse (Fv-4~“) were thymectomized at about 4 weeks. The surface of thymocytes from individual progeny was labeled with i”I, and then the cell lysates were immune precipitated with anti-G4W serum (Lot 41ml) (lane l), with anti-gp70 serum (lane 2), and with anti-H@ serum (lane 3). The precipitates were analyzed by electrophoresis on 10% polyacrylamide gel. Ten days after thymectomy, the mice were injected intraperitoneally with FLV (1.9 X 10’ PFU and 1.5 X lo3 FFU). Ten days later they were killed, and the individual spleens were weighed and homogenized to be tested for LLV. In general, we scored animals as susceptible to FLV when their spleens weighed more than 0.4 g.
0.16
Spleen wt.(g)
30K
80K 73K
NO.1 NO-2 123123123123123123123
?s $ ’
136
IKEDA
AND
brane gp7Os were detected on FRG thymocytes by absorption tests similar to those in Fig. 3 (data not shown). They were indistinguishable in molecular weight and antigenicity from the 80K and 73K antigens of C4W mice. AKR mice expressed two size classes of membrane gp7Os which were demonstrated in absorption test (Fig. 5). The larger gp70 (77K) appeared to be serologically related, but not identical, to the 80K antigen of C4W mice since the 77K antigen was not recognized by “Fv-4 type-specific” antiserum (Table 2). The smaller gp70 (72K) was serologically indistinguishable from the 73K antigen of C4W thymocytes. It should be stressed that the 77K antigen of AKR mice has the same serological reactivity as ecotropic viral gp7Os in our same assay system. The %3K and 8OK antigens of C4 W thymocytes were glycoproteins. To determine whether the 73K and 80K antigens of C4W thymocytes, though antigenically related to MuLV gp70, are glycosylated proteins, we analyzed the lectin-binding capacity of the two antigens. Since Con A specifically binds cy-D-manopyranoside or a-D-glucopyranoside, Con A-Sepharose 4B was mixed with the lysate of iodinated C4W thymocytes and washed with lysing buffer. The 73K and 80K antigens were not detected in the supernatant, but successfully eluted from the Con A-Sepharose 4B with a-methyl-D(+)-glucoside-containing lysing buffer (data not shown). This suggests that the Fv-.&‘-associated 73K and 80K molecules were glycoproteins. DISCUSSION
This comparative study between BALB/ c and its Fv-4 congenic C4W suggests that Fv-$-mediated resistance is associated with the expression of MuLV gp70-related cell surface antigens. Several distinct classes of antigens were identified on thymocytes from C4W mice with molecular weights of approximately 80K, 73K, and 15K. The 80K and 73K proteins were found to be glycosylated and serologically related to MuLV gp70. Genetic experiments demonstrated that the 80K antigen seg-
ODAKA
-94
-68 -45
FIG. 5. Sequential immune precipitation analysis of iodinated surface proteins from AKR/J thymocytes. (A) Three identical lysates of ‘%I-labeled AKR/ J thymocytes were immune precipitated either with anti-Fv-4w’” serum (Lot 85) (A lane l), with anti-G serum (Lot 1210), (B lane l), with anti-RLV serum (C lane 1). The immune complexes were cleared by adding fixed S. aureus and centrifuging. Each of the unprecipitated supernatants was further immuneprecipitated with anti-RLV serum (A lane 2), with anti-RLV serum (B lane 2), or with anti-G serum (lot 1210) (C lane 1). The immune precipitates were analyzed by electrophoresis on 10% polyacrylamide gels.
regated with Fv-Q-controlled resistance to FLV infection. A quantitative difference in the expression of the 73K antigen was detected between this congenic pair, but this difference was not associated with resistance in the backcross mice (Fig. 4). Thus, the Fv-4 gene may represent structural information for a unique cell surface gp70 or a regulatory locus which governs expression of this gp70. The dominant resistant allele at the Fv4 locus confers resistance to infection of FLV and ecotropic MuLVs (Kai et al, 1976; Odaka et al, 1981), and also appears to specify the expression of membrane gp70.
Fv-l
GENE
AND
CELL
The Fv-4 gene is thought to influence certain viral replication steps specific for ecotropic MuLV (Kai et a& 1976; Yoshikura et ul, 19’79; Odaka et aL, 1981). In the first step of virus infection, ecotropic and amphotropic MuLVs bind individually the specific receptors of mouse cells, respectively controlled by the Real gene (chromosome 5) (Oie et aZ., 1978; Ruddle et aL, 1978; Marshall and Rapp, 1979) and RumI gene (chromosome 8) (Gazdar et al, 1977), although these specific receptors have yet to be defined. The receptor binding component of MuLV is the major envelope glycoprotein gp70 (Gazdar et uk, 1977; Oie et al., 1978; Ruddle et al., 1978; Marshall and Rapp, 1979). Thus, the most simple hypothesis we can envision for Fv-h-mediated resistance is a viral interference model: in Fv-&resistant mice the specific receptor on cell membranes to ecotropic MuLVs might be already bound by the Fv4’-controlled gp7Os, and therefore the binding of exogenous ecotropic MuLVs is inhibited. This model also could explain the Fv-4’-mediated suppression of endogenous ecotropic MuLV expression in hybrids of AKRxFRG (Odaka et CL& 1980), because, if the endogenous AKR proviruses are activated in the hybrid mice, their secondary spread to surrounding cells is obstructed. This proposed mechanism for Fv-Q-mediated resistance is not unique; other systems have been described in which resistance can be correlated with the expression of viral gp7Os. The avian genes h-E, ev3, and ev6 control both cellular expression of the env gene of the endogenous subgroup E provirus and host resistance to exogenous subgroup E virus infection (Robinson and Lamoreux, 1976; Ando and Toyoshima, 1976; Robinson et al, 1981). The resistance of several mouse strains to Friend MuLV-induced erythroproliferative disease was correlated with the endogenous expression of an MCF/xenotropic virus gp70-related proteins on the surface of cells of resistant mice (Ruscetti et al, 1981; Bassin et uL, 1982). Using the Tung classification of the cell membrane gp7Os (Tung et aL, 1978; see Table 2), our data suggest that the Fv-4’as-
MEMBRANE
gp’70
137
sociated 73K protein is antigenically related to GIx-gp70 of 129 mice or 0-gp7O of B6 mice, while the 80K antigen is a new type of membrane gp70, but probably closely related to ec+-gp70 of AKR mice (Table 2). Several other gp70-related cell surface antigens (e.g., GIx (Stockert et uL, 1971), G~RADAI) (Obata et cd, 19W, G(AKSL~) (Stockert et al, 1979) GcEmn) (Obata et al, 1981), and XenCSA (Morse et &, 1979) have been identified on mouse lymphocytes by cytotoxic assay or membrane immunofluorescence using specific antisera. Though we have not directly compared these antigens with the Fv-$‘-associated 80K antigen, the strain distributions of these other antigens are clearly distinct from that of the Fv-J-linked antigen (Table 2). ACKNOWLEDGMENTS We thank K. Kai, H. Yoshikura, and the Division of Cancer Cause and Prevention, NCI, for providing us with antisera; S. Suzuki, K. Suzuki, and T. Noguchi for providing us with inbred strains of mice; M. Yamashita, K. Asahi, and Y. Kagami for technical assistance; H. Sato, Y. Sanai, and F. Taguchi for reading this manuscript and helpful discussion; and N. Munezawa for preparation of the manuscript. We are grateful to people at the National Institutes of Health, especially C. A. Kozak, W. Y. Langdon, and H. C. Morse, III for their correction of English of this manuscript and S. Grove for the preparation of the manuscript. This research was supported in part by a grant from Japanese Ministry of Education and by a grant from the Mitsubishi Foundation.
REFERENCES ANDO, T., and TOYSHIMA, K. (1976). Genetic control of chick helper factor in cells which lack natural group-specific antigen of avian leukosis. VirdoQy 73.521-52’7. BASSIN, R. H., RUSCETTI, S., ALI, I., HAAPALA, D. K., and REIN, A. (1982). Normal DBA/Z mouse cells synthesize a glycoprotein which interferes with MCF virus infection. Virology 123, 139-151. BASSIN, R. H., TUNE, N., and FISCHINGER, P. J. (1971). Rapid cell culture assay technique for murine leukaemia viruses. Nature &n&m) 229,5644X6. CULLEN, S. E., FREED, J. H., and NATHENSON, S. G. (1976). Structural and serological properties of murine Ia alloantigens. Transplant. Rev. 29,236-270. GARDNER, M. B., RASHEED, S., PAL, B. K., Ems, J. D., and O’BRIEN, S. J. (1980). Ahn-I, a dominant murine leukemia virus restriction gene, is poly-
138
IKEDA
morphic in leukemia-prone Acad
Sci
AND ODAKA
wild mice. Proc. Nat.
USA 77,531-535.
GAZDAR, A. F., OIE, H., LALLEY, P., Moss, W. W., and MINNA, J. D. (1977). Identification of mouse chromosomes required for murine leukemia virus replication. CeU 11, 949-956. IKEDA, H., SATO, H., and ODAKA, T. (1981). Mapping of the Fv-.4 mouse gene controlling resistance to murine leukemia viruses. Ink J. Cancer 28,2X7-240. KAI, K., IKEDA, H., YUASA, Y., SUZUKI, S., and ODAKA, T. (1976). Mouse strain resistant to N-, B-, and NBtropic murine leukemia viruses. J. Vird 20, 436440.
LAEMMLE, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-687. MARSHALL, T. H., and RAPP, U. R. (1979). Genes controlling receptors for ecotropic and xenotropic type C virus in Mus ceruicolor and Mus musculus. J. Virol 29,501-506. MORSE III, H. C., CHUSED, T. M., BOEHM-TRUITT, M., MATHIESON, B. J., SHARROW, S. O., and HARTLEY, J. W. (1979). XenCSA: Cell surface antigens related to the major glycoproteins (gp70) of xenotropic murine leukemia viruses. J. Immurwl 122.443-454. OBATA, Y., STOCKERT, E., O’DONNEU, P. V., OKUBO, S., SNYDER,H. W. JR., and OLD, L. J. (1978). GcmAn: A new cell surface antigen of mouse leukemia defined by naturally occurring antibody and its relationship to murine leukemia virus. J. Exp. MecL 147.1089-1105. OBATA, Y., STOCKERT, E., DELEO, A. B., O’DONNELL, P. V., SNYDER, H. W. JR., and OLD, L. J. (1981). G (aRLn):A cell surface antigen of the mouse related to xenotropic MuLV defined by naturally occurring antibody and monoclonal antibody. J. Exp. Med 154.659-675. ODAKA, T., and MATSUKURA, M. (1969). Inheritance of susceptibility to Friend mouse leukemia virus. VI. Reciprocal alteration of innate resistance or susceptibility by bone marrow transplantation between congenic strains. J. Viral. 4, 337-343. ODAKA, T., IKEDA, H., MORIWAKI, K., MATSUZAWA, A., MIZUNO, M., and KONDO, K. (1978). Genetic resistance in Japanese wild mice (Mus musculus molossinus) to an NB-tropic Friend murine leukemia virus. J. Nat. Cancer Inst. 61,1301-1306. ODAKA, T., IKEDA, H., and AKATSUKA, T. (1980). Restricted expression of endogenous N-tropic XCpositive leukemia virus in hybrids between G and AKR mice: An effect of the Fv-l’ gene. Int. J. Can~~-25.757-762.
ODAKA, T., IKEDA, H., YOSHIKURA, H., MORIWAKI, K., and SUZUKI, S. (1981). Fv-6: Gene controlling resistance to NB-tropic Friend murine leukemia virus. Distribution in wild mice, introduction into genetic background of BALB/e mice, and mapping of chromosomes. J. Nat. Cancer fnst 67,1123-1127.
OIE, H. K., GAZDAR, A. F., LALLEY, P. A., RUSSELL, E. K., and MINNA, J. D. (1978). Mouse chromosome 5 codes for ecotropic murine leukaemia virus eellsurface receptor. Nature (London) 274, 60-62. OLD, L. J., and STOCKERT, E. (1977). Immunogenetics of cell surface antigens of mouse leukemia. Annzl Rev. Genet. 11, 127-160. ROBINSON, H. L., and LAMOREUX, W. F. (1976). Expression of endogenous ALV antigens and susceptibility to subgroup E ALV in three strains of chickens (Endogenous avian C-type virus). viralogy 69,50-62. ROBINSON, H. L., ASTRIN, S. M., SENIOR, A. M., and SALAZAR, F. H. (1981). Host susceptibility to endogenous virus: Defective, glycoprotein-expressing proviruses interfere to infections. .J. viral. 40,745751. ROWE, W. P., PUGH, W. E., and HARTLEY, J. W. (1970). Plaque assay techniques for murine leukemia viruses. virology 42, 1136-1139. RUDDLE, N. H., CONTA, B. S., LEINWAND, L., KOZAK, C., RUDDLE, F., BESMER, P., and BALTIMORE, D. (1978). Assignment of the receptor for ecotropic murine leukemia virus to mouse chromosome 5. J. Exp. Med 148,451-465. RUSCE~, S., DAVIS, L., FE&D, J., and OLIFF, A. (1981). Friend murine leukemia virus-induced leukemia is associated with the formation of mink cell focusinducing viruses and is blocked in mice expressing endogenous mink cell focus-inducing xenotropic viral envelope genes. J. Exp. Med 154, 907-920. SILVER, J., CECKA, J. M., MCMILLAN, M., and HOOD, L. (1976). Chemical characterization of products of the H-2 complex. Cold Spring Harbor Symp. Quad. Biol. XLI, 369-377. STOCKERT,B., OLD, L. J., and BOYSE, E. A. (1971). The GIx system. A cell surface allo-antigen associated with murine leukemia virus: Implications regarding chromosomal integration of the viral genome. J. Exp. Med 133,1334-1335. STOCKERT,E., DELEO, A. B., O’DONNELL, P. V., OBATA, Y., and OLD, L. J. (1979). GCAKSUj:A new cell surface antigen of the mouse related to the dualtropic mink cell focus-inducing class of murine leukemia virus detected by naturally occurring antibody. J. Esp. Med.
149,200-215.
SUZUKI, S. (1975). FV-4: A new gene affecting the splenomegaly induction by Friend leukemia virus. Japan. J. Exp. Med 45.473-478. SUZUKI, S., and MATSUBARA, S. (1975). Isolation of Friend leukemia virus resistant line from noninbred mouse colony. Japan. J. Exp. Med 45,467471. TUNG, J. S., SHEN, F. W., FLEISSNER, E., and BOYSE, E. A. (1976). X-gp70: A third molecular species of the envelope protein gp70 of murine leukemia virus, expressed on mouse lymphoid cells. J. Exp. Med
143,969-974.
Fv-4 GENE
AND CELL
TUNG, J. S., O’DONNELL, P. V., FLEISSNER, E., and BOYSE, E. A. (1978). Relationships of gp70 of MuLV envelopes to gp’70 components of mouse lymphocyte plasma membranes. J. Exp. Mea! 147, 12801284. WRIGHT, B. S., O’BRIEN, P. A., SHIBLEY, G. P., MAYYASI, S. A., and LASFARGUES, J. C. (1967). Infection of an established mouse bone marrow cell line (JLS-V9) with Rauscher and Moloney murine leukemia viruses. Cancer Res. 27,1672-1677. YOSHIDA, M., and YOSHIKURA, H. (1981). A glycoprotein specified by spleen focus-forming virus in three
MEMBRANE
gp70
139
cell lines varies in molecular size and peptide composition, but retains both xenotropic and ecotropic MuLV antigenicity. J. Gen ViroL 52, 183-188. YOSHIKURA, H., NAITO, H., and MORIWAKI, K. (19’79). Unstable resistance of G mouse fibroblasts to ecotropic murine leukemia virus infection. J. ViroL 29, 1078-1086. YOSHIKURA, H., and ODAKA, T. (1982). Surface antigen expressed in hematopoietic cells derived from Fv-4’ mouse strains. J. Nut. Cancer Inst. 68, 10051009.