Rauscher MuLV ts mutants and xenotropic type ... including recombinants rec 25b-14, rec 25c-3, and rec .... deoxythymidylate[oligo(dT)]-cellulose (type 3 from.
JOURNAL OF VIROLOGY, July 1981, p. 219-228
Vol. 39, No. 1
0022-538X/81/070219-10$02.00/0
Recombinants Between Temperature-Sensitive Mutants of Rauscher Murine Leukemia Virus and BALB:Virus-2: Genetic Mapping of the Rauscher Murine Leukemia Virus Genome JOSEPH MERREGAERT,t MARIANO BARBACID, AND STUART A. AARONSON* Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20205 Received 28 July 1980/Accepted 27 March 1981
Recombinant viruses were generated in tissue culture between Rauscher murine leukemia virus (MuLV) temperature-sensitive (ts) mutants restricted at different steps in virus replication and a mouse endogenous xenotropic virus, BALB:virus2. Mutants used included ts 28, a late mutant which releases noninfectious viruses at 390C, and ts 29, a double mutant with a ts lesion in its reverse transcriptase and a late block affecting virus budding. Immunological typing of the translational products of clonal recombinant viruses made it possible to establish their partial genetic maps and localize regions of the viral genome affected by different ts lesions. Recombinants involving Rauscher MuLV ts 28 invariably contained BALB:virus-2 p15, p12, and p30 proteins, localizing the late defect in replication by this mutant to the 5' moiety of the viral gag gene. All ts 29-derived recombinants contained the entire BALB:virus-2 gag and pol genes. Substitution of the pol gene is in agreement with the reported thermolability of Rauscher MuLV ts 29 reverse transcriptase (Tronick et al., J. Virol. 16:1476-1482, 1975). Substitution of the gag gene suggests that intemal structural proteins are actively involved in the virus budding processing. Rauscher MuLV recombinants were used to establish the genetic map of the Rauscher MuLV genome by T, oligonucleotide fingerprinting analysis. Detection of Rauscher MuLV T, oligonucleotides in representative recombinant viruses, whose protein phenotypes were established by immunological techniques, permitted their assignment to specific regions of the viral genome. The genetic map of Rauscher MuLV generated in these studies should be useful for identifying and characterizing the viral gene(s) involved in leukemogenesis.
Replication-competent murine type C viruses C viruses in an effort to map the viral gene(s) induce tumors of hemopoietic cells. Investiga- involved in functions required for replication tion of these agents has led to the demonstration and leukemogenesis (1). The present report deof the existence of at least three discrete viral scribes the generation of recombinants between genes, gag, pol, and env, which code for the core the xenotropic endogenous BALB:virus-2 and structural proteins, the reverse transcriptase, Rauscher MuLV conditional mutant ts 28 or ts and the envelope proteins, respectively (3). It is 29 which are restricted at different steps in the not known yet whether these or as yet uniden- viral replicative cycle (32, 33, 37, 40, 41). Immutified viral genes are directly involved in virus- nological analysis of the recombinant viruses induced tumorigenesis. Our laboratory has been generated has made it possible to localize regions involved in investigating replication and trans- in each mutant responsible for different blocks forming functions of a clonal isolate of Rauscher to virus replication. In addition, these recombimurine leukemia virus (MuLV). This virus in- nants have allowed us to construct a detailed T, duces tumors of B-lymphoid cells (27), in con- oligonucleotide map of the Rauscher MuLV getrast to mouse leukemia viruses such as AKR or nome. Moloney MuLV that induce T-cell lymphomas MATERIALS AND METHODS (13,22, 24). Cell culture. Cells were grown in Dulbecco's modWe have recently developed a tissue culture system for generating recombinants between ification of Eagle minimal essential medium suppleSerum Co., Rauscher MuLV ts mutants and xenotropic type mented with 10% calf serum (Coloradoclonal lines of Denver, Colo.). The cells used included t On leave from the Department of Radiobiology of the continuous mouse cells BALB/3T3 and NIH/3T3 (17). A cell line derived from an embryo culture of a wild Studiecentrum voor Kernenergie, Mol, Belgium. 219
220
MERREGAERT, BARBACID, AND AARONSON
mouse (WM-C) from a colony maintained by M. Collins, Microbiological Associates, Bethesda, Md., has been described (1). Viruses. A clonal isolate of Rauscher MuLV and temperature-sensitive (ts) mutants of this virus, ts 28 and ts 29, have been described (32). An inducible xenotropic endogenous virus, BALB:virus-2, was originally isolated after 5-iodo-2'-deoxyuridine induction of BALB/c embryo cells in tissue culture (2). Recombinants of Rauscher MuLV ts 25 and BALB:virus-2, including recombinants rec 25b-14, rec 25c-3, and rec 25e-8 (1), and an in vivo-generated xenotropic host range recombinant of Rauscher MuLV, D17-3B3 (6), have been reported. Infection of cells was performed according to previously published methods (2). Type C virus synthesis by infected cultures was measured by an assay for virion-associated reverse transcriptase activity in tissue culture fluids (29). Typing radioimmunoassays for MuLV proteins. The isolation of the p15, p12, p30, and gp7O structural proteins of Rauscher MuLV and BALB: virus-2 as well as the reverse transcriptase and the gag gene-coded plO proteins of Rauscher MuLV have been described in detail (4, 7, 15, 20, 23, 36). Each protein was labeled with 125I to high specific activity by the chloramine-T method (12) and used, along with caprine anti-Rauscher MuLV and BALB:virus-2 sera, to develop appropriate type-specific competition ra-
dioimmunoassays. Typing of recombinant pl5E viral proteins. The highly hydrophobic properties of the env genecoded pi5E (16, 30) have made it very difficult to develop type-specific radioimmunoassays for this protein. The origin of pi5E was determined by the different electrophoretical mobilities of Rauscher MuLV pl5E and BALB:virus-2 pl5E proteins. Cells infected with parental or recombinant viruses were labeled with [3S]methionine as previously described (5), and their postmicrosomal cell extracts were immunoprecipitated with a caprine antiserum elicited against autologous cells infected with BALB:virus-2 which contained high-titered anti-pl5E antibodies. Immunoprecipitates, collected with the aid of Staphylococcus aureus protein A bound to Sepharose-4B beads (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.), were analyzed by electrophoresis in 6 to 12% linear sodium dodecyl sulfate (SDS)-polyacrylamide gradient gels as described elsewhere (5). The electrophoretical mobility of the pl5E protein encoded by each recombinant virus (whether identical to that of Rauscher MuLV pl5E or to that of BALB:virus p15E) determined the parental origin of the viral genomic region coding for this protein. Isolation of viral RNA. 32P-labeled MuLV RNA was prepared from supernatant fluids harvested from virus-infected cultures as described by Coffin and Billeter (8). 32P-labeled virus was disrupted in 1 ml of 10 mM Tris-hydrochloride (pH 7.8), 100 mM NaCl, and 1 mM EDTA (TNE buffer) containing 1% SDS and incubated with proteinase K (Calbiochem) (1 mg/ml) at 300C for 30 min. The digestion mixture, supplemented with 2 mg of carrier RNA, was extracted with an equal volume of a 1:1 mixture of redistilled phenol (buffered with TNE) and chloroform-isoamyl alcohol (24:1); the phenol phase was reextracted with an equal
J. VIROL.
volume of TNE buffer. The combined aqueous phases were extracted once more with phenol-chloroformisoamyl alcohol (25:24:1) and twice with chloroform. RNA was precipitated by the addition of 0.2 volume of 1 M sodium acetate (pH 5.0) and 2.5 volumes of ethanol and stored at -20°C. To prepare 70S RNA, the ethanol precipitate was dissolved in TNE buffer, incubated for 15 min at 30°C with proteinase K (0.5 mg/ml), and centrifuged on a 10 to 30% sucrose gradient in TNE in a Beckman SW41 rotor at 40,000 rpm for 3 h at 4°C. The specific activity of the RNA was about 5 x 106 cpm/,tg. Fingerprinting and characterization of RNase TL-resistant oligonucleotides. Viral 70S RNA was digested with RNase T, (Calbiochem, Los Angeles, Calif.) at a ratio of 1 U per 10 fig of RNA for 45 min at 370C and analyzed by two-dimensional gel electrophoresis as described by De Wachter and Fiers (9). The first dimension was run in a 10% polyacrylamide gel at pH 3.5 in the presence of 6 M urea, and the second dimension was run in a 22% polyacrylamide gel at pH 8.0. RNase T, oligonucleotides were eluted from the gels and completely digested with RNase A (Worthington Biochemicals Corp., Freehold, N.J.), and the digestion products were identified on polyethyleneimine thin-layer plates (Polygram Cel 300; 20 by 20 cm; Macherey-Nagel and Co., Diiren, West Germany)
(39). T1 oligonucleotide fingerprints of the extreme 3' end of the viral genome were prepared as follows. Lowmolecular-weight RNA from the 70S gradient was pooled, and the polyadenylate [poly(A)+]-containing fragments were isolated by chromatography on oligodeoxythymidylate[oligo(dT)]-cellulose (type 3 from Collaborative Research, Inc., Waltham, Mass.) (see below). Poly(A)+-containing fragments were centrifuged on a 5 to 20% sucrose gradient in TNE containing 0.1% SDS in a Beckman SW41 rotor at 40,000 rpm for 3.5 h at room temperature. RNA of approximately 5S was pooled and fingerprinted as described above. Oligonucleotide mapping. Oligonucleotide mapping was performed as described (8). 32P-labeled 70S RNA was partially degraded by heating the sample dissolved in TE buffer (10 mM Tris-hydrochloride [pH 7.8] and 1 mM EDTA) for 30 s in a boiling water bath and cooling on ice. RNA samples were made 0.5 M LiCl and 0.5% SDS, allowed to enter an oligo(dT)cellulose column (0.2-mi bed volume), and washed with 15 ml of high-salt buffer (500 mM LiCl, 10 mM Tris-hydrochloride [pH 7.8], and 0.5% SDS). Bound RNA was eluted with low-salt buffer (10 mM LiCl, 10 mM Tris-hydrochloride [pH 7.8], and 0.5% SDS). By making the eluate 0.5 M LiCl, RNA recovered in the first cycle could be recycled without an intervening ethanol precipitation step. Poly(A)'-tagged RNA fragments, isolated by two cycles of binding to oligo(dT)cellulose, were precipitated by addition of 200 jig of carrier RNA, 1 volume of 0.4 M sodium acetate (pH 5.0), and 2 volumes of ethanol. The poly(A)+-containing fragments were dissolved in 0.4 ml of TNE buffer plus 0.1% SDS and layered on top of a 10-ml 5 to 20% linear sucrose gradient in TNE buffer containing 0.1% SDS. Sedimentation was performed in a Spinco SW41 rotor at 40,000 rpm for 3.5 h at room temperature. RNA fragments differing from each other by 5 to 10S
VOL. 39, 1981
RAUSCHER MuLV RECOMBINANTS
increments were pooled and precipitated with 2 volof ethanol after addition of 200 ug of RNA. This RNA was then completely hydrolyzed with RNase T1, and the digestion products were separated by twodimensional gel electrophoresis as described above.
umes
TABLE 1. Frequency of recombinants between Rauscher MuLV ts 28 or ts 29 and BALB:virus-2' Virus titer (PIU/ml) at following temperature
(00)
Virus source
RESULTS
In vitro generation of recombinants between Rauscher MuLV ts mutants and BALB:virus-2. We have reported (1) the isolation of recombinants between ts 25, a mutant of Rauscher MuLV blocked in gag gene precursor cleavage (34), and the endogenous xenotropic BALB:virus-2 (2). At 390C on NIH/3T3 celLs, replication of ts mutant and xenotropic parental viruses was effectively blocked, whereas recombinants possessing Rauscher MuLV envelope functions and BALB:virus-2 sequences in gag gene sequences affected by the ts lesion grew efficiently. A wild mouse embryo cell line, WM-C, permissive for BALB:virus-2 replication, was chronically infected with this virus and then superinfected with either Rauscher MuLV ts 28 or ts 29 at the permissive temperature (310C). ts 28 represents a physiological class which releases noninfectious viruses at 390C (32), whereas ts 29 is a double mutant with a defect in reverse transcriptase and a late block affecting virus budding (37, 41). To control for mutant leakiness or reversion, WM-C cells, infected with either mutant alone, were passaged under identical conditions. After 2 weeks, virus released from each culture was tested for infectivity for NIH/3T3 cells at the restrictive temperature. If the infectivity of the virus at 390C was significantly enhanced by passage of the mutant through WM-C cells replicating the xenotropic virus, individual virus clones were selected by the microtiter procedure for further analysis. Table 1 demonstrates the results of several experiments designed to generate recombinant viruses. Both ts 28 and ts 29 were stable, exhibiting no evidence of infectivity at 390C after propagation for 2 weeks at 310C in WM-C cells. However, after passage in BALB:virus-2-producing WM-C cells, virus infectivity for NIH/ 3T3 cells at the nonpermissive temperature ranged from 100°5 to 1015 polymerase-inducing units/ml compared with 104-5 to 1055/ml at 310C. Virus growth at 390C could have resulted from reversion of the mutant to wild type or from complementation or recombination with the xenotropic virus. Thus, assuming that all of the viruses capable of propagation at the nonpermissive temperature were true recombinants, the frequency of recombinants, as determined from the ratio of infectivity at the two temperatures, would be 0.003 to 0.03% (Table 1).
221
31
WM-C cells +BALB:virus-2 +Wild-type Rauscher MuLV +ts 28 +ts 29
39
39/31
4U] A2G 23 4 [7C, 2(AC), 2(AU), A2U, 6U] G [C., 2(AC), A40, Uy] AG 24 5 [9C, AC, A2C, 5U] G [C,, Uy, A2C, A2U, A5U] GC 25 6 [8C, AC, A2C, AU, 4U] A2GC [2C, AC, A2C, A2U, 4U] AG 26 7 [7C, 2(AC), A3C, A2U, 3U] G [3C, 2(AC), A2C, 2(AU), 2U] G 27 8 [4C, 3(AC), 2(A2C), A2U, 2U] AG [4C, 2(AC), 2(AU), 4U] AG 28 9 [5C, 2(AU), A2U, 2(AC), 4U] G [2C, AC, A2C, A3U, 3U] G 29 10 [6C, 2(AC), 2(A20), U3] G [3C, 2(AU), A3U, 5U] AG 30 11 [6C, 2(AC), 2(A2WC), U2] G [3C, AC, 6U] G 31 12 [6C, AC, A2X, AU, A4U, 2U] G [3C, AC, AU, 6U] AG 32 13 [8C, 2(AC), AU, 4U] G [4C, AC, 3(AU), U] G 33 14 [2C, AC, A2U, A3U] A4G [3C, 2(AU), A2U, 5U] G 34 15 [5C, AU, A2U, A3U, 4U] Gd [2C, 2(AC), A2C, A5C, 3U] A2G 35 16 [10C, AC, AU, A2U, 4U] G [2C, A2C, A2U, U] AG 36 17 [5C, AC, AU, A3U, A4U, 2U] AG [3C, AU, A2U, 2U] G 37 18 [7C, AC, A2C, 5U] G [2C, AC, A40, 2U] AG 19 [8C, 2(AC), AU, U2] AG Numbers refer to oligonucleotides diagrammed in Fig. 2. These oligonucleotides were reproducibly detected in complete T, digestion fingerprints of different preparations of 70S Rauscher MuLV. b Compositions were determined by quantitation of the secondary digestion products. All data are from the average of three or more determinations and were rounded to the nearest integer. 'Oligonucleotides 5 and 6 were only poorly resolved. Composition of 6 was derived from poly(A)+-containing fragments missing spot 5. Pancreatic compositions of oligonucleotides 30, 35, 36, and 37 were derived from analysis of spots derived from short poly(A)-containing fragments (see Fig. 3). Analysis of oligonucleotides 19, 20, and 23 was hampered by contamination from the oligonucleotides migrating just above. C, and Uy denote that the amounts of cytidylic and uridylic residues remained undetermined. d The sequence of oligonucleotide 15 has been deduced from the recently reported Rauscher MuLV "strong stop" sequence as: (G) UAUCCAAUAAAUCCUCUUG (21). The underlined sequence was confirmed by partial nuclease PI digestion of 5'-32P-end-labeled oligonucleotide 15 after electrophoresis and homochromatography (data not shown).
Rauscher MuLV. In conjunction with the partial genetic analysis obtained by phenotypic characterization of each of the known viral translational products, we were able to associate regions of the oligonucleotide map with genetic functions. Oligonucleotides with map positions 2 to 9 segregated with the gag region. For example, these oligonucleotides were present in rec 25b14, which synthesized Rauscher MuLV translational products p15, p12, and p30, and were completely missing in rec 25c-3, 28b-21, 29a-3, and 29d-10, which possessed BALB:virus-2-derived gag gene proteins. Oligonucleotide 3 (map position 10) was found to segregate with the viral gag gene-coded plO, since it was present in rec 28b-21 but not in rec 25b-14. Oligonucleotides 28, 14, 29, and 12 seemed to be associated with coding sequences for the Rauscher MuLV reverse transcriptase (Fig. 4). Rec 25c-3 and rec 29d-10 were indistinguishable by immunological criteria. However, the T, fingerprint of rec 29d-10 showed an additional Rauscher-derived T, oligonucleotide 6. From Fig. 4 it is apparent that the crossing over which separated pol and env regions was located in the
neighborhood of oligonucleotide 6. It was not possible to map this oligonucleotide in eitherpol or env regions. Thus, it may reside within the intercistronic region. The detailed mapping of oligonucleotides associated with the Rauscher MuLV env gene was largely due to the analysis of rec 29a-3 and D173B3, an in vivo-isolated recombinant from a Rauscher MuLV-infected BALB/c mouse (6). These oligonucleotides were shown to be located in the 3' half of the viral RNA molecule between map positions 16 and 31. Rec 29a-3, the only recombinant with a pl5E protein derived from BALB:virus-2, defined the boundary between gp7O- and p15E-coding regions of the env gene near oligonucleotide 19. This recombinant apparently originated by a double cros&sover, one in the 5' part of the env region, since Rauscher MuLV oligonucleotides 2 and 8 were missing, and the other near the 3' end of gp7O-coding sequences. D17-3B3 was the only recombinant possessing BALB:virus-2 gp7O-specific antigenic determinants. This virus lacked at least Rauscher MuLV oligonucleotides 2, 7, 8, 10, and 17 in the env gene. These findings suggest that
VOL. 39, 1981
this recombinant arose as a result of recombination between Rauscher MuLV and BALB:virus-2, most likely within the 5' end region of the env gene. Sequences nearest the 3' end of the viral genome have been terned the "c" region. This region includes the genomic terminal redundant sequence which contains around 70 nucleotides in Moloney MuLV. Our studies do not allow a precise definition of the boundaries of env and
RAUSCHER MuLV RECOMBINANTS
225
"c" regions. However, a sequence corresponding to that of oligonucleotide 21 was found by Sutcliff et al. (35) within the Moloney MuLV env gene reading frame beyond the pl5E-coding sequence. This helps to define the "c" region of Rauscher MuLV between map positions 32 and 36. The fact that oligonucleotide 15 was located within the terminal redundant sequence provided an excellent genetic marker for that region. T1 fingerprints of all recombinants were pre-
FIG. 3. T, RNA fingerprints of fragmented poly(A)+-containing Rauscher MuLV RNA obtained from fractions containing 2 to 5S (A), 6 to 10S (B), 11 to 16S (C), and 17 to 26S (D) RNA. By visual inspection, it is apparent that oligonucleotides lying farther from the 3' end of the genome faded in intensity in fingerprints of smaller size classes of poly(A) -selected RNA. For example, oligonucleotides 9 and 19 gradually faded in fingerprints C and D and were completely missing in B.
226
ro. JJ. VIROL.
MERREGAERT, BARBACID, AND AARONSON
H
r-
"Pc"~-reion tr
env
PO/
gag
Vrs
p15E
gp7O
plO Rev. Trarscrip.
p30
p15 p12
3,
5, 5 27 26 18
15 (24 16 13 1
oligonucleotkiden0:
+
1+)1(+
3)1(2814 2912) 6 ?
+++++.
?
?
.
.
2 8 (7 10 17 23) 9 31 19 34 30 25 32 4 36 21 33 37 35 11 15 + + + + +)+
.
.
+
+(+)(+) + +(+)+
+
+
+
+
++
+
mU
m~~
rec 25b- 14:
26 27 28 29 30 31 32 33 34 35 36
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
1
Map Position:
rec 25c-3:
.+ . . + W~~~ ~~
.
+
+
+
+ 1+) + (44 +
+
1+fl+)(+
.
+
.
+
+
+0
+
++
rec 25e-8: ++. .
.
.
.
.
.
.
+
+ 1+
1+) +++
rec 28b-21:
++ (40 1+) + (+ 1+
rmc 29o-3:
rmc 29d-10: +
.
.
.+1+1 +
+
+
+
+
+ . .
+
.
?
+
+
+ (+( +(+)(+fl
+
+
+
H
DI,r383: 3
of Rauscher MuL V. The physical map of large T, oligonucleotides of Rauscher MuL V of the figure. tJligonucleotides 20 and 22 (Table 2) have not been mapped. The presence of each Rauscher MuL V oligonucleotide in a given recombinant virus is indicated by +. Different + symbols were assigned based upon the type of analysis performed: +, identity of a Rauscher MuL V oligonucleotide was confirmed by quantitative analysis of its secondary digestion products; (+), oligonucleotide did not possess a unique electrophoretic mobility and its secondary digestion products were contaminated by those of a second comigrating T1 oligonucleotide; +, oligonucleotide was confirmed by oligonucleotide was present at the corresponding electrophoretic position, but its qualitative analysis; secondary digestion products were not distinctive enough to allow identification. Proteins derived from FIG. 4. Genetic map
70S RNA is shown in the upper part
Rauscher MuL V
(ci)
or
BALB:virus 2
(m) parental
viruses
pared from short poly(A)'-containing fragments, as
described
found in
regions
in
Materials
cases
all
were
and
Methods.
that the 5'- and
derived from
only
one
We
3'-terminal
of the paren-
tal viruses. A second observation of interest is that among the crossovers
seven
recombinants, four had
very close to the 3' terminus. Those
are
indicated. trs, Terminal redundant sequence.
parental viruses, it was possible to isolate frequencies ranging from to 0.03%. Recombinant viruses generated
of both
recombinant viruses at 0.003
between
BALB:virus-2
and
ts
28,
a
mutant
particles at 3900 (32, 41), invariably demonstrated antigenic determinants of the 5' moiety of the BALB:viruswhich releases noninfectious
recombinants contained
2 gag gene. These results localize the ts lesion
not 15. Whether the
affecting
bination observed in
genome. In each case, recombinants
oligonucleotide 11, but high frequency of recointhis region pertains in some
way to the mechanism involved in viral
bination
or
recoin-
simply the selection for reenv region of the viral genome
reflects
combinants in the
remains to be determined.
DISCUSSION This report describes the isolation and char-
ts 28 to the gag
region of the viral involving
Rauscher MuLV and ts 29 substituted the verse
transcriptase
re-
of BALB:virus-2 for that of
findings confirm preindicating that the ts 29 reverse transcriptase has a ts lesion affecting its enzymatic functions (37). ts 29 has also been reported Rauscher MuLV. These
vious studies
to
possess
budding
a
second
lesion that affects virus
nonperinissive temperature (33, fact that all recombinants involving ts
at the
acterization of recombinant viruses between ts
41). The
mutants of Rauscher MuLV restricted at differ-
29 contained the gag gene of BALB:virus-2 sug-
ent steps in virus replication and a xenotropic endogenous mouse type C virus, BALB:virus-2. By taking advantage of the growth restrictions
gests that products of this region of the viral genome may be
budding
process.
actively
involved in the virus
VOL. 39, 1981
Rauscher MuLV ts 28 appears to process its in normal fashion (38). In contrast, a ts lesion at the carboxy terminus of the viral gag gene blocks normal processing of the gag gene product in Rauscher MuLV ts 25 (1). Rauscher MuLV ts 29 has also been reported to exhibit impaired gag gene processing at the nonpermissive temperature (38). Thus, lesions affecting the gag gene can result in several readily distinguishable phenotypic blocks to virus replication. By oligonucleotide fingerprinting analysis of Rauscher MuLV and several of the recombinant viruses isolated here and previously (1), it was possible to construct an oligonucleotide map of Rauscher MuLV. Oligonucleotides with map positions 2 to 10 segregated with the gag region, helping to confirn the location of that region of the genome near the 5' end of the molecule (10, 11). Oligonucleotides with map positions 11 to 14 corresponded with the pol gene and linked gag to pol. This order is consistent with the detection of precursor polyproteins containing both gag and pol gene products of Rauscher MuLV in vivo (18) and with in vitro translation studies of Moloney and Rauscher MuLV RNA (19, 25). T1 oligonucleotides at positions 16 to 24 corresponded with gp7O-coding sequences, locating the env gene toward the 3' end of the molecule. This agrees with the genome location for gp7O previously reported for MuLV's that have arisen by recombination within their respective env genes (14, 28, 31). Our system for the generation of recombinants selected for viruses with the mouse cell-tropic host range of the Rauscher MuLV parent. The conservation of Rauscher MuLV gp7O in all recombinants analyzed provides strong support for the concept that this gene product is solely responsible for the ecotropic host range of Rauscher MuLV. Each recombinant virus, independent of its genotype, grew at a similar efficiency in NIH/3T3 cells in tissue culture. This was particularly striking in the case of rec 29a-3, whose parental Rauscher MuLV contribution was only in sequences coding for gp7O. Thus, it appears that endogenous xenotropic mouse type C viruses, if provided with the appropriate gp7O, can replicate in mouse cells as efficiently as oncogenic exogenous viruses. The mechanism by which replication-competent type C viruses cause leukemia is still poorly understood. These viruses replicate in fibroblasts in tissue culture without causing transformation, but are capable of causing various forms
gag gene precursor
of leukemia in their natural hosts. The clone of Rauscher MuLV characterized in this study has been shown to induce tumors with characteristics of pre-B-lymphoid cells (27). Analysis of the
RAUSCHER MuLV RECOMBINANTS
227
leukemogenicity of different recombinant viruses isolated and characterized in the present studies should be useful in determining what regions of the Rauscher MuLV genome are essential for its oncogenic functions and its tropism for transformation of a specific lymphoid cell target. ACKNOWLEDGMENTS We thank Angela Galen, Linda Long, and Christian Thomae for excellent assistance.
LITRATURE CITED 1. Aaronson, S. A., and M. Barbacid. 1980. Viral genes involved in leukemogenesis. I. Generation of recombinants between oncogenic and non-oncogenic mouse type C-viruses in tissue culture. J. Exp. Med. 151:467480. 2. Aaronson, S. A., and J. R. Stephenson. 1973. Independent segregation of foci for activation of biologically distinguishable RNA C-type viruses in mouse cells. Proc. Natl. Acad. Sci. U.S.A. 70:2055-2058. 3. Baltimore, D. 1975. Tumor viruses: 1974. Cold Spring Harbor Symp. Quant. Biol. 39:1187-1200. 4. Barbacid, M., and S. A. Aaronson. 1978. Membrane properties of the gag gene-coded p15 protein of mouse type C RNA tumor viruses. J. Biol. Chem. 253:14081414. 5. Barbacid, M., A. V. Lauver, and S. G. Devare. 1980. Biochemical and immunological characterization of polyproteins coded for by the McDonough, Gardner-Amstein, and Snyder-Theilen strains of feline sarcoma virus. J. Virol. 33:196-207. 6. Barbacid, M., K. C. Robbins, S. Hino, and S. A. Aaronson. 1978. Genetic recombination between mouse type-C RNA viruses: a mechanism for endogenous viral gene amplifcation in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 75:923-927. 7. Barbacid, M., J. R. Stephenson, and S. A. Aaronson. 1976. Structural polypeptides of mammalian type CRNA viruses: isolation and immunologic characterization of a low molecular weight polypeptide plO. J. Biol. Chem. 251:4859-4866. 8. Coffin, J. M., and M. A. Bflleter. 1976. A physical map of the Rous sarcoma virus genome. J. Mol. Biol. 100: 293-318. 9. De Wachter, R., and W. Fiers. 1972. Preparative twodimensional polyacrylamide gel electrophoresis of 3Plabeled RNA. Anal. Biochem. 49:184-197. 10. Faller, D. G., and N. Hopkins. 1978. T, oligonucleotide maps of N-, B-, and B-*NB-tropic murine leukemia viruses derived from BALB/c. J. Virol. 26:143-152. 11. Faller, D. G., and N. Hopkins. 1978. T, oligonucleotides that segregate with tropism and with properties of gp70 in recombinants between N- and B-tropic murine leukemia viruses. J. Virol. 26:153-158. 12. Greenwood, F. C., W. M. Hunter, and J. S. Clover. 1963. The preparation of "3'I-labeled human growth hormone of high specific radioactivity. Biochem. J. 89: 114-123. 13. Gross, L. 1951. "Spontaneous" leukemia developing in C3H mice following inoculation in infancy with AKleukemic extracts or AK-embryos. Proc. Soc. Exp. Biol. Med. 76:27-32. 14. Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. Rowe. 1977. A new class of murine leukemia virus associated with development of spontaneous lymphomas. Proc. Natl. Acad. Sci. U.S.A. 79:789-792. 15. Hino, S., J. R. Stephenson, and S. A. Aaronson. 1976. Radioimmunoassays for the 70,000 molecular weight
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