Vol. 23, No. 1 Printed in U.S.A.
JOURNAL OF VIROLOGY, JU1y 1977, p. 188-195 Copyright X) 1977 American Society for Microbiology
RNase Ti-Resistant Oligonucleotides of B-Tropic Murine Leukemia Virus from BALB/c and Five of Its NB-Tropic Derivatives DOUGLAS V. FALLER AND NANCY HOPKINS* Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received for publication 10 March 1977
We used two-dimensional gel electrophoresis to obtain fingerprints of RNase Tl-resistant oligonucleotides of a B-tropic murine leukemia virus from BALB/c and five NB-tropic viruses independently derived from this B virus by passage through NIH Swiss mouse embryo cells in vitro. The fingerprints of the B- and NB-tropic viruses were very similar: approximately 33 of 35 large Ti-resistant oligonucleotides appeared to be shared by these viruses. However, the five NBtropic viruses possessed an apparently common alteration relative to their B virus progenitor. This change involved the acquisition of one oligonucleotide and, tentatively, the loss of one oligonucleotide. We do not know whether these changes represent an alteration responsible for the change from B- to NBtropism. Fingerprints of B- and NB-tropic viruses were not affected when the viruses were grown in cells of different Fv-i type.
Naturally occurring ecotropic type C RNA viruses can be classified according to their host range as either N- or B-tropic depending upon whether they grow preferentially on cells derived from NIH Swiss or BALB/c mice, respectively (6). Laboratory strains of mice resemble either NIH Swiss or BALB/c in their relative sensitivity to N- and B-tropic viruses. This sensitivity is determined by the Fv-1 gene (16, 19). NIH Swiss mice are designated Fv-in; BALB/c mice are Fv-ib. F1 hybrids of Fv-ln and Fv-lb strains are resistant to both N- and B-tropic viruses. The Fv-1 gene plays a role in blocking leukemogenesis by murine leukemia viruses of incompatible host range (see reference 17). Although the mechanism of Fv-i restriction is not known, it appears that viral infection of a cell of incompatible Fv-1 type is blocked after absorption and penetration but before integration of proviral DNA (11, 13, 14, 21). The viral genes that determine N- or B-tropism have not yet been identified, although recent evidence suggests that these putative genes code for products that are present in virions (20). Fv-i restriction of a virus of incompatible tropism is relative rather than absolute (6). Usually, the progeny of an infection of N cells with B-tropic virus or B cells with N-tropic virus retain their original tropism. Sometimes, however, forced passage of a virus through Fv-1 incompatible cells results in the selection of viruses with stably extended host range, designated NB-tropic, that grow well on cells from
Fv-1ln, Fv-lb, and Fv-lnb mice (6; see reference 17). NB-tropic viruses have been obtained from B-tropic viruses after infection of Fv-ln mouse embryo cells in vitro (J. Hartley, T. Pincus, and W. Rowe, 1971, unpublished data [see reference 17]; 8). The conversion from N- to NB-tropism has been accomplished in vivo (17). The genetic basis for the change in tropism is not known. We were interested in determining the relatedness of the genomes of NB-topic viruses to those of their B-tropic virus progenitors. One method of genome analysis that has proved useful with type C viruses of chickens is analysis of the set of large oligonucleotides obtained after RNase Ti digestion of 32P-labeled viral RNA (4, 22). Thus, we have analyzed the Tiresistant oligonucleotides, after separation on two-dimensional polyacrylamide gels, termed "fingerprints," of the RNA of B-tropic virus derived from the BALB/c mouse and of five NBtropic viruses independently derived from this B virus in three different laboratories by serial passage of the B virus on NIH Swiss mouse embryo cells.
MATERIALS AND METHODS Viruses. B-tropic viruses included: WN1802B (6), which was obtained from T. Pincus, B-clone 11 and B-clone 18 (12) from P. Jolicoeur, and B-clone E. S. from E. Scolnick. NB-tropic viruses included: WN1802BN'B-clone 1, which was cloned from WN1802BN'B, an NB-tropic virus derived from WN1802B (J. Hartley, T. Pincuis, and W. Rowe, unpublished data; see 17), which was obtained from 188
VOL. 23, 1977
RNase Ti-RESISTANT OLIGONUCLEOTIDES
189
J. Hartley. NB-18-1-clone 5 and NB-11-3-clone 1 (8, digested with 15 U of RNase T1 (Calbiochem) for 30 9) have been described. NB-E.S.-clone 744 and NB- min at 37°C. After digestion, the mixture was adE.S.-clone 745 were obtained from E. Scolnick. justed to 6 M urea with solid ultrapure urea Cells, media, and virus infections. The BALB/ (Schwarz/Mann), and a dye mixture containing 2 3T3, NIH/3T3, and Sc-i (7) cells, media, and method mg each of xylene cyanol FF and bromphenol blue of virus infections have been described (8). Chroni- per ml, 50% (wt/wt) sucrose, and 6 M urea was cally infected cells (infected at least several weeks added. The two-dimensional gel electrophoresis was before use) were used to prepare 32P-labeled virus. carried out by the method of DeWachter and Fiers To obtain B-tropic virus grown in Fv-ln cells, NIH/ (5). The first dimension was run in a 10% acrylamide 3T3 was infected with B-clone-18 at a multiplicity of gel at pH 3.5 and in the presence of 6 M urea, and infection of approximately 5. After 4 months in cul- the second dimension was run in 21.8% acrylamide ture the cells were cloned, and a cell line that pro- at pH 8.0. duced virus that gave plaques (-105 PFU/ml) on Extraction and characterization of oligonucleoBALB/3T3 but not on NIH/3T3 cells was used as a tides. After electrophoresis, the oligonucleotides source of B-tropic virus. were located by autoradiography and cut out with Preparation of 32P-labeled RNA. Confluent cul- an 8-mm-diameter cork borer. Gel disks were placed tures of chronically infected cells were incubated for in tubes containing 0.5 ml of water and 25 ,g of 4 h in phosphate-free Dulbecco-modified Eagle me- carrier RNA, and their radioactivity was quantidium containing 10% dialyzed calf serum and then tated by Cerenkov counting. After elution for 48 h at for 12 h in medium of the same composition but con- 4°C, 60 to 90% of the radioactivity could be found in taining 1 mCi of [32P]phosphate (New England Nu- the eluate. Gel disks were washed with 0.3 ml of clear Corp.) per ml. Medium was then replaced with water, and the combined eluates were lyophilized. Dulbecco-modified Eagle medium containing 20% After digestion with pancreatic RNase A (Worthingcalf serum. This medium was harvested at four 8- to ton), the oligonucleotides were analyzed as de12-h intervals, clarified by low-speed centrifugation, scribed by Adams et al. (1). and stored at -20'C. Virus was pelleted, and the Digestion with penicillium nuclease P1 (Yamasa pellets were suspended in 50 mM Tris-hydrochloride Biochemical, Tokyo) was for 1 h at 37°C in 10 mM (pH 7.5)-100 mM NaCl-1 mM EDTA (TNE buffer) Tris-hydrochloride, pH 6, at an enzyme-to-RNA and adjusted to 125 gg of carrier RNA (Sigma grade ratio (wt/wt) of 1:100. Base composition of total viral VI yeast RNA, repurified by phenol extraction) per RNA and of primary or secondary oligonucleotides ml and 1% sodium dodecyl sulfate. was done by digestion at 37°C for 30 min with a mixThis mixture was extracted two or three times ture of Takadiastase (Calbiochem; Sanzyme-R) (18) with equal volumes of a 1:1 mixture of redistilled and RNase A as described by Flanegan et al. (in phenol (buffered with TNE and chloroform-isoamyl press). The products from P1 or Takadiastase-RNase alcohol [24:1]), and the RNA was precipitated by A digestion were separated by electrophoresis on addition of 2 volumes of ethanol and storage at Whatman 3 MM paper at pH 3.5 (2). After digestion -20'C. The method of Flanegan et al. (J. B. Flane- and electrophoresis, the products were located by gan, R. F. Pettersson, V. Ambros, M. J. Hewlett, autoradiography and quantitated by cutting out the and D. Baltimore, Proc. Natl. Acad. Sci. U.S.A., in spots and counting them in a xylene-based scintilpress) was used to establish that this protocol labels lation fluid (Econofluor, New England Nuclear the individual nucleotides with equal specific activi- Corp.). ties. RESULTS To prepare 70S RNA, the ethanol precipitate Rowe and Hartley (6) have described the isowas dissolved in 10 mM Tris-hydrochloride (pH 7.5)100 mM NaCl-1 mM EDTA-0.5% sodium dodecyl lation of B-tropic virus from BALB/c mice by sulfate (SDS buffer) and centrifuged on a 15 to 30% serial passage of an extract of the spleen of nonsucrose gradient in SDS buffer in a Beckman SW41 diseased adult BALB/c mice through BALB/c rotor at 36,000 rpm for 2.5 h at 20°C. mouse embryo cells in vitro. One such isolate, 35S RNA was prepared by dissolving ethanol- designated WN1802B, has served as a prototype precipitated 70S RNA in 10 mM Tris-hydrochloride B-tropic virus for many studies and has been (pH 7.5)-i mM EDTA-0.5% SDS, heating to 100°C for 30 s, and quick-cooling on ice. This heat-dena- cloned in several laboratories. We infected tured RNA was centrifuged on a 15 to 30% sucrose BALB/3T3 cells with WN1802B and each of gradient as described above for 5 h. Also, 35S RNA three clonal isolates, B-clone 18, B-clone 11, was prepared by the method of Leis (15) with a 5 to and B-clone E.S., derived from WN1802B. Fin20% sucrose gradient containing 80% dimethyl sulf- gerprints obtained by two-dimensional gel elecoxide. RNA sequences containing polyadenylic acid trophoresis of the RNase Ti-resistant oligonuregions were selected by chromatography on poly- cleotides generated after digestion of 32P-lauridylic acid-Sephadex as described by Coffin and beled 70S RNA from each of these four viruses Billeter (4). of the Two-dimensional electrophoresis. RNA samples are shown in Fig. 1A to D. A diagram in 2. of B-clone-E.S. shown is fingerprint Fig. were adjusted to a total of 100 ug with carrier RNA and were dried on a polyethylene sheet. Digestion As expected, the fingerprints of these four Band electrophoresis were carried out essentially as tropic viruses are very similar, although two of described by Billeter et al. (3). Briefly, samples were the clones, B-clone 11 and B-clone E.S., possess
190
FALLER AND HOPKINS
J. VIROL.
one oligonucleotide that is not present in WN1802B, and B-clone E.S. also lacks one ohgonucleotide relative to WN1802B (see arrows in Fig. 1B and D). That the arbitrarily numbered oligonucleotides seen in Fig. 2 are derived from the viral genome is based on two lines of evidence. (i) The same pattern of oligonucleotides was obtained for NB-E.S.-clone 745 after digestion of either 70S RNA, 35S RNA obtained by denaturation of 70S RNA and sedimentation in 80% dimethyl sulfoxide gradients, or polyadenylic acid-selected 35S RNA (data not shown). (ii) The numbered oligonucleotides, with the exception of B-35, are present in approximately molar amounts relative to one another. Because the second dimension of the fingerprint separates oligonucleotides primarily on the basis of size, the relative molarity of the oligonucleotides can be determined by quantitating the
radioactivity present in each "spot." Further digestion of each Ti-resistant oligonucleotide with pancreatic RNase or complete digestion to mononucleotides and quantitation of the digestion products allows sizing of the Ti-resistant oligonucleotides and thus allows more accurate quantitation of their molarity. Table 1 lists the pancreatic RNase digestion products and the nucleotide length (±+ 20%) of numbered oligonucleotides of B-clone E.S. The oligonucleotide numbered B-35 in Fig. 2 appears "darker" than the two flanking oligonucleotides in the fingerprints of the B-tropic viruses (Fig. 1A-D), suggesting that this "spot" is a "doublet" consisting of two oligonucleotides that co-migrate. Quantitation of the radioactivity present in this "spot" also implies that it is present in twicemolar amount. Further analysis of spot 35 (and likewise the flanking spots 27 and 28) has been complicated by the proximity of the many unre-
10W
,qw
w
w ...-
4~~~~ ... a
0
v
4b6
--'
*
w~~~
46
0
0
0
0
B WN 1802B
A
p
9
Ar~
S
0
0
a
a
0 0~ a
NB E
I-i
WNI802BN B Clone
i-..
1.
i.-,
:". f:.
I
f, ':
F
FIG. 1. Two-dimensional gel electrophoretic fingerprints of 32P-labeled RNase Ti-resistant oligonucleotides of B- and NB-tropic viruses. B-tropic viruses: (A) WN1802B, (B) B-clone 11, (C) B-clone 18, (D) B-clone E.S. Arrows in (B) and (D) indicate oligonucleotides "unique" to B-clone 11 and B-clone E.S., respectively. Box surrounds region that is altered in NB-tropic viruses. NB-tropic viruses: Each NB-tropic virus was derived from the B-tropic virus pictured directly above it. (E) WN1802BN'B-clone 1, (F) NB-11-3-clone 1, (G) NB-18-1-clone 5, (H) NB-E.S.-clone 744, (I) NB-E.S.-clone 745. Arrows in (F and G) indicate oligonucleotides that are not present in the B-tropic virus progenitors of NB-11-3-clone 1 and NB-18-1-clone 5, respectively. The box in each figure is drawn around the region in which there is a change in all five NB-tropic viruses relative to their B-virus progenitors: one oligonucleotide is gained and one appears to be lost in this region.
VOL. 23, 1977
RNase Ti-RESISTANT OLIGONUCLEOTIDES
solved oligonucleotides migrating slightly ahead of it. Preliminary analysis of the products obtained after pancreatic RNase digestion of B-35 reveals the presence of U, C, (AG), (AAC), and several (AC)'s. Attempts to separate the B-35 doublet into its presumed component oligonucleotides, including electrophoresis on DEAE-cellulose at pH 1.9-7% formic acid, have been unsuccessful. We next infected BALB/3T3 cells with each of five NB-tropic viruses derived from the four Btropic viruses analyzed above. The origin of
191
these viruses is summarized in Fig. 3. NBtropic viruses derived from different clones of B-tropic virus or from cloned versus uncloned WN1802B must have arisen independently of one another. NB-E.S.-clone 744 and clone 745 were both derived from B-clone E.S.; however, these viruses have been shown to differ: when the virion proteins of these viruses and of Bclone E.S. are examined on SDS-polyacrylamide gels, NB-E.S.-clone 744 and clone 745 are found to possess a p30 that migrates ahead of the B virus p30 but, in addition, NB-E.S.-clone
a
0 a
0
0
S.
'a
*5
05 0aS
0
S
B
B
B-Clone 18
C
B- Clone ES
D a
9
dZ
51
4
S
0
1%
*
50
S _a
*
0 0
w
0
NB G
NB
NB-1lB--Clone 5
NB- ES- Clone 744
H 'S
SA 'S* S
%
I FIG. 1. C, D, G, H and
9 I.
NB NB-ES-Clone 745
192
FALLER AND HOPKINS
J. VIROL.
FIG. 2. Diagrammatic representation of fingerprints of B-clone E.S. and NB-E.S.-clone 744 or 745. Numbered oligonucleotides are those whose pancreatic RNase digestion products have been analyzed. In the five NB-tropic viruses, there is a new oligonucleotide, NB-34, relative to the B-tropic virus progenitors, and spot 35 is a single oligonucleotide rather than a doublee" as in the B-tropic viruses. Arrows indicate direction of migration in first and second dimensions of the gel electrophoresis. XC and B indicate position of dye markers xylene cyanol FF and bromphenol blue, respectively.
745 possesses a p15 with altered electrophoretic mobility relative to the p15 of B-clone E.S., suggesting that these two NB-tropic viruses are independent isolates (9; J. Schindler, R. Hynes, and N. Hopkins, unpublished data). Figures 1E to I show the fingerprints of the five NB-tropic viruses derived from the B viruses pictured directly above them. The NBtropic virus fingerprints look very similar to those of their B-tropic virus progenitors. This apparent similarity is further supported by the fact that (numbered) oligonucleotides that appear to migrate similarly between B-clone E.S. and NB-E.S.-clone 745 yield the same products after digestion with pancreatic RNase (Table 1).
Although the B- and NB-tropic viruses apparently share 33 to 34 of 35 to 36 large T1resistant oligonucleotides, each NB-tropic virus possesses an apparently common alteration relative to its B-tropic virus progenitor. This alteration, which is found in the region of the fingerprints enclosed by the box and is shown closeup in Fig. 4, involves the acquisition of a new oligonucleotide, designated NB-34 in Fig. 2, in each NB-tropic virus. It apparently also in-
volves the loss of one oligonucleotide from the doublet, B-35, found in each B-tropic virus, since NB-35 appears to show an intensity equal to that of the two flanking oligonucleotides. That the new oligonucleotide, NB-34, is probably the same in all five viruses is suggested by the fact that the products of pancreatic RNase digestion of NB-34 are the same for the five viruses. Analysis of oligonucleotide NB-35, like that of B-35, is hampered by contamination from the oligonucleotides migrating just above. However, the pancreatic RNase digestion products of NB-35 consist of U, C, (AG), and (AAC), and a loss of several (AC)'s relative to B-35 is apparent. The pancreatic RNase digestion products of NB-35 appear to be the same for all five NBtropic viruses. It is interesting to note the possibility of a relationship between the hypothetical oligonucleotide, which has been lost at position 35 in the NB-tropic viruses, and the new oligonucleotide, which appears in these viruses at position NB-34. NB-34 possesses several (AC's and ends in AG. Since NB-35 has lost several (AC)'s relative to B-35, it is conceivable that one oligonucleotide in B-35 has been al-
VOL. 23, 1977
RNase Ti-RESISTANT OLIGONUCLEOTIDES
TABLE 1. RNase Ti-resistant oligonucleotides of Bclone E.S. and its NB-tropic derivatives Oligonucleotide
Chain length (nucleotides)c
Composition"
no. a
1
5U, 8C, 4(AC), 2(AU).
2
2(A2C), G 10U, 15C, (AU), (A3C), (A4C), G 2U, 7C, (AU),
3
\2C) -.,
(AXC), (AXC),
32
+-
20%
40
(A2G)
4
5
6 7 8 9 110 111 112
1U, 5C, (AC), (AU), (A2U), (A5C) G 1U, 6C, (AC), 2(AU), (A2U), (A4C) G 2U, 5C, 2(A2C), (A2U), (AG) 5U, 10C, 2(AC), G 4U, 7C, 2(AC), 3(AU), (AG) 6U, 5C, 3(AC), (AU), (A2C), G 2U, 13C, 3(AC), (A3C), G 3U, 12C, 2(AC), (A3C), (AG) 1U, 4C, 3(AC), (A2U), (A4G) 2U, 8C, 2(AC), (A2C), G 1U, 2C, 2(A2C), (A5C), (AG) 5U, 6C, 2(A2U), (A5U), G 7U, 8C, 2(AC), (AU), G 8U, llC, G 4U, 3C, (AC), 2(AU), (A4G) 4U, 5C, (AC), (AU), (A2U), (AG) 4U, 6C, 2(AC), 2(AU), G 3U, 3C, 3(AU), (A2C), G 5U, 2C, 2(AC), (A2U), G 6U, 3C, (AC), (A2G) G 5U, 4C, (AC), 3(AU) G 4U, 3C, (AU), (A2U) G 5U, 1C, 2(AU), (AG)
193
tered to generate NB-34. However, since oligonucleotides with similar composition will have similar electrophoretic mobilities in the first dimension, the fact that NB-34 and the "postulated" oligonucleotide apparently possess common pancreatic RNase digestion products does not necessarily imply a relationship between these two oligonucleotides. In addition to the two common changes, two of the five NB-tropic viruses, NB-11-3-clone 1 and NB-18-1-clone 5, each possess a new, "unique" oligonucleotide relative to their B-virus progenitors (see arrows, Fig. 1F and G). We have examined the fingerprints of Bclone 18, B-clone E.S., NB-18-1-clone 5, and NB-E.S.-clone 744 viruses grown in Sc-1 cells (a cell line that does not show Fv-1 restriction), NB-11-3-clone 1 grown in NIH/3T3 cells, and Bclone 18, which had grown in NIH/3T3 cells but retained its B-tropism. The fingerprints showed the same pattern of oligonucleotides as those of the corresponding viruses grown in BALB/3T3 cells.
DISCUSSION Fingerprints of the RNase Ti-resistant oligonucleotides of a B-tropic virus of BALB/c and five of its NB-tropic derivatives were shown to .5 24 be similar. Approximately 33 of 35 large very 16 22 Ti-resistant oligonucleotides appeared to be L7 20 shared by these viruses. However, all five NBL8 18 tropic viruses were found to possess a common L9 18 alteration relative to their B-virus progenitor. This alteration involves the acquisition of one 10 19 oligonucleotide and, probably, the loss of one 211 16 oligonucleotide. Preliminary results are not in212 15 consistent with the possibility that the new 213 15 "NB-oligonucleotide" could have been gener214 18 ated by an alteration in the oligonucleotide that 2 S5 13 has disappeared relative to the B-tropic virus; 216 12 2U, 8C, 2(AC), (A2C), however, the data on this point are far from 19 19 2 (AG) conclusive. Any number of mechanisms-sin10 2U, 9C, 3(AC), (AG) 19 gle base change, deletion, insertion, or recom3 11 3U, 5C, 3(AC), (A5C), G 21 bination with a different endogenous virus3 12 1U, 7C, (AC), (A2C), 18 could generate the alterations in the RNase T13 (A3C), G resistant oligonucleotides that we have ob13 8U, 1C, (AC), (A2U), G 15 served in the NB-tropic viruses. NB-314 2U, 2C, 4(AC), (A2U), 17 A question of obvious interest is whether (AG) these alterations reflect a change(s) that is rea Numbers refer to oligonucleotide spots diasponsible for the change from B- to NB-tropism. gramed in Fig. 2. If homologous spots of B-clone We cannot answer this question at present. The E.S. and its NB-tropic derivatives have the same facts that this change occurred in five viruses composition, they are listed only once. The oligonu- isolated in three different laboratories, that the cleotide that is present in NB-E.S.-clones 744 and fingerprints appear to be unaltered when the 745 but not in B-clone E.S. is designated NB-34. b Compositions are determined by quantitation of NB-tropic viruses are grown in NIH/3T3, 113 114
18 17
the products of pancreatic RNase digestion. Average values for at least four determinations are given and are rounded to the nearest integer.
c Chain length was computed by addition of the nearest integers of the molar yields.
194
J. VIROL.
FALLER AND HOPKINS WN1802B B-clone 11
B-clone 18
WN1802BN'B-clone 1
B-clone E.S.
NB-E.S.-clone 744
NB-18-1-clone 5
NB-E.S.-clone 745
NB-11-3-clone 1
FIG. 3. Origin of B- and NB-tropic viruses. B-tropic viruses: WN1802B (6) is an uncloned B-tropic virus derived by serial passage of the extract of a spleen of a non-diseased adult BALBIc mouse through secondary BALBIc embryo cells; B-clone 18 (12), B-clone 11 (12), and B-clone E.S. (E. Scolnick, unpublished data) are clonal isolates derived from WN1802B. NB-tropic viruses: WN1802BN'B-clone 1 is a clonal isolate derived from WN1802BN'B (J. Hartley, T. Pincus, W. Rowe, unpublished data) derived from WN1802B by serial passage on secondary NIH Swiss embryo cells; NB-18-1-clone 5 (9), NB-11-3-clone 1 (9), NB-E.S.-clone 744 (E. Scolnick, unpublished data) and NB-E.S.-clone 745 (E. Scolnick, unpublished data) are clonal isolates derived from B-clone 18, B-clone 11, and B-clone E.S., respectively, by serial passage through NIHI3T3 cells.
: Do
*
B
_NB
FIG. 4. Close-up of the region of the fingerprints in which NB-tropic viruses differ from their B-tropic virus progenitors. (B) Close-up of region surrounded by a box in Fig. IA (B-tropic virus WN1802B). (NB) Close-up of this region from Fig. lE (NB-tropic virus WN1802BN'B-clone 1).
BALB/3T3, or Sc-1 cells, and that a B-tropic virus grown in NIH/3T3 cells, which retained its B-tropism, did not show the alterations observed in the NB-tropic viruses are at least consistent with the possibility that the changes observed may be related to the genetically stable change to NB-tropism. We are currently attempting to "map" the new oligonucleotide present in the NB-tropic viruses relative to the 3' end of the genome (4, 22). Although the gene order of murine leukemia viruses is not yet known, it seems quite likely that it will be similar to that of the avian type C viruses. Furthermore, we are currently analyzing the RNase Ti-resistant oligonucleotides of an N-tropic virus of BALB/c and 21 recombinants between this virus and the Btropic virus, B-clone 18, described here (8). Since these recombinants appear to possess different combinations of N- and B-virus virion proteins (10; J. Schindler, R. Hynes, and N. Hopkins, unpublished data), this analysis may
yield a map order for these proteins. Hopkins et al. (9) have found that the NB-tropic viruses analyzed here possess an altered p30 relative to their B-tropic virus progenitors. It will be interesting to see whether the new NB-oligonucleotide lies in a region of the genome where the gag gene, which codes for p30 and other internal virion proteins, is expected to reside. ACKNOWLEDGMENTS We thank John Coffin for a first-hand demonstration of this method of RNA fingerprinting and for very helpful discussions. We thank John Schindler for analyzing the proteins of NB-E.S.-clone 744 and 745 viruses. We thank Paul Gottlieb and Phillip Sharp for helpful criticisms. This work was supported by Public Health Service research grants from the National Cancer Institute (CA 19308 to N. H., and CA 14051 to the Center for Cancer Research, Massachusetts Institute of Technology).
ADDENDUM IN PROOF Preliminary determinations of the relative order of the large Ti-resistant oligonucleotides of NB-ESclone 745 with respect to the 3' end of the genome
VOL. 23, 1977
RNase Ti-RESISTANT OLIGONUCLEOTIDES
indicate that oligonucleotide NB-34 is among the four oligonucleotides closest to the 5' end. LITERATURE CITED 1. Adams, J. M., P. G. N. Jeppersen, F. Sanger, and B. G. Barrell. 1969. Nucleotide sequence from the coat protein cistron of R17 bacteriophage RNA. Nature (London) 223:1009-1014. 2. Barrell, B. G. 1971. Fractionation and sequence analysis of radioactive nucleotides, p. 751-758. In G. L. Cantoni and D. R. Davies (ed.), Procedures in nucleic acid research, vol. II. Harper and Row, New York. 3. Billeter, M. A., J. T. Parsons, and J. M. Coffin. 1974. The nucleotide sequence complexity of avian tumor virus RNA. Proc. Natl. Acad. Sci. U.S.A. 71:35603564. 4. Coffin, J. M., and M. A. Billeter. 1976. A physical map of the Rous sarcoma virus genome. J. Mol. Biol. 100:293-318. 5. DeWachter, R., and W. Fiers. 1972. Preparative twodimensional polyacrylamide gel electrophoresis of 32P-labeled RNA. Anal. Biochem. 49:184-197. 6. Hartley, J. W., and W. P. Rowe. 1975. Host-range restriction of murine leukemia viruses in mouse embryo cell cultures. J. Virol. 5:221-225. 7. Hartley, J. W., and W. P. Rowe. 1975. Clonal cell lines from a feral mouse embryo which lack -host-range restrictions for murine leukemia viruses. Virology 65:128-134. 8. Hopkins, N., P. Traktman, and K. Whalen. 1976. Ntropic variants obtained after co-infection with Nand B-tropic murine leukemia viruses. J. Virol. 18:324-331. 9. Hopkins, N., J. Schindler, and R. Hynes. 1977. Six NBtropic viruses derived from a B-tropic virus of BALB/c have altered p30. J. Virol. 21:309-318. 10. Hopkins, N., J. Schindler, and P. Gottlieb. 1977. Evidence for recombination between N- and B-tropic murine leukemia viruses. J. Virol. 21:1074-1078. 11. Huang, A., P. Besmer, L. Chu, and D. Baltimore. 1973. Growth of pseudotypes of vesicular stomatitis virus with N-tropic murine leukemia virus coats in cells resistant to N-tropic viruses. J. Virol. 12:659-662.
195
12. Jolicoeur, P., and D. Baltimore. 1975. Effect of the Fv-1 locus on the titration of murine leukemia viruses. J. Virol. 16:1593-1598. 13. Jolicoeur, P., and D. Baltimore. 1976. Effect of Fv-1 gene product on proviral DNA formation and integration in cells infected with murine leukemia viruses. Proc. Natl. Acad. Sci. U.S.A. 73:2236-2240. 14. Krontiris, T. G., R. Soeiro, and B. N. Fields. 1973. Host restriction of Friend leukemia virus. Role of the viral outer coat. Proc. NatI. Acad. Sci. U.S.A. 70:25492553. 15. Leis, J. P. 1976. RNA-dependent DNA polymerase activity of RNA tumor virus. VI. Processing mode of action of avian myeloblastosis virus polymerase. J. Virol. 19:932-939. 16. Lilly, F. 1967. Susceptibility of two strains of Friend leukemia virus in mice. Science 155:461-462. 17. Lilly, F., and T. Pincus. 1973. Genetic control of murine viral leukemogenesis. Adv. Cancer Res. 17:231277. 18. Niramaru, M., T. Uchida, and F. Egami. 1966. Ribonuclease preparation for the base analysis of polyribonucleotides. Anal. Biochem. 17:135-142. 19. Pincus, T., J. W. Hartley, and W. P. Rowe. 1971. A major genetic locus affecting resistance to infection with murine leukemia viruses. I. Tissue culture studies of naturally occurring viruses. J. Exp. Med. 133:1219-1233. 20. Rein, A., S. V. S. Kashmiri, R. H. Bassin, B. I. Gerwin, and G. Duran-Troise. 1976. Phenotypic mixing between N- and B-tropic murine leukemia viruses: infectious particles with dual sensitivity to Fv-1 restriction. Cell 7:373-379. 21. Sveda, M. M., and R. Soeiro. 1976. Host restriction of Friend leukemia virus: synthesis and integration of the provirus. Proc. Natl. Acad. Sci. U.S.A. 73:23562360. 22. Wang, L., D. Galehouse, P. P. Mellon, P. Duesberg, W. S. Mason, and P. K. Vogt. 1976. Mapping oligonucleotides of Rous sarcoma virus RNA that segregate with polymerase and group-specific antigen markers in recombinants. Proc. NatI. Acad. Sci. U.S.A. 73:3952-3956.
JOURNAL OF VIROLOGY, JU1y 1977, p. 188-195 Copyright X) 1977 American Society for Microbiology
RNase Ti-Resistant Oligonucleotides of B-Tropic Murine Leukemia Virus from BALB/c and Five of Its NB-Tropic Derivatives DOUGLAS V. FALLER AND NANCY HOPKINS* Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received for publication 10 March 1977
We used two-dimensional gel electrophoresis to obtain fingerprints of RNase Tl-resistant oligonucleotides of a B-tropic murine leukemia virus from BALB/c and five NB-tropic viruses independently derived from this B virus by passage through NIH Swiss mouse embryo cells in vitro. The fingerprints of the B- and NB-tropic viruses were very similar: approximately 33 of 35 large Ti-resistant oligonucleotides appeared to be shared by these viruses. However, the five NBtropic viruses possessed an apparently common alteration relative to their B virus progenitor. This change involved the acquisition of one oligonucleotide and, tentatively, the loss of one oligonucleotide. We do not know whether these changes represent an alteration responsible for the change from B- to NBtropism. Fingerprints of B- and NB-tropic viruses were not affected when the viruses were grown in cells of different Fv-i type.
Naturally occurring ecotropic type C RNA viruses can be classified according to their host range as either N- or B-tropic depending upon whether they grow preferentially on cells derived from NIH Swiss or BALB/c mice, respectively (6). Laboratory strains of mice resemble either NIH Swiss or BALB/c in their relative sensitivity to N- and B-tropic viruses. This sensitivity is determined by the Fv-1 gene (16, 19). NIH Swiss mice are designated Fv-in; BALB/c mice are Fv-ib. F1 hybrids of Fv-ln and Fv-lb strains are resistant to both N- and B-tropic viruses. The Fv-1 gene plays a role in blocking leukemogenesis by murine leukemia viruses of incompatible host range (see reference 17). Although the mechanism of Fv-i restriction is not known, it appears that viral infection of a cell of incompatible Fv-1 type is blocked after absorption and penetration but before integration of proviral DNA (11, 13, 14, 21). The viral genes that determine N- or B-tropism have not yet been identified, although recent evidence suggests that these putative genes code for products that are present in virions (20). Fv-i restriction of a virus of incompatible tropism is relative rather than absolute (6). Usually, the progeny of an infection of N cells with B-tropic virus or B cells with N-tropic virus retain their original tropism. Sometimes, however, forced passage of a virus through Fv-1 incompatible cells results in the selection of viruses with stably extended host range, designated NB-tropic, that grow well on cells from
Fv-1ln, Fv-lb, and Fv-lnb mice (6; see reference 17). NB-tropic viruses have been obtained from B-tropic viruses after infection of Fv-ln mouse embryo cells in vitro (J. Hartley, T. Pincus, and W. Rowe, 1971, unpublished data [see reference 17]; 8). The conversion from N- to NB-tropism has been accomplished in vivo (17). The genetic basis for the change in tropism is not known. We were interested in determining the relatedness of the genomes of NB-topic viruses to those of their B-tropic virus progenitors. One method of genome analysis that has proved useful with type C viruses of chickens is analysis of the set of large oligonucleotides obtained after RNase Ti digestion of 32P-labeled viral RNA (4, 22). Thus, we have analyzed the Tiresistant oligonucleotides, after separation on two-dimensional polyacrylamide gels, termed "fingerprints," of the RNA of B-tropic virus derived from the BALB/c mouse and of five NBtropic viruses independently derived from this B virus in three different laboratories by serial passage of the B virus on NIH Swiss mouse embryo cells.
MATERIALS AND METHODS Viruses. B-tropic viruses included: WN1802B (6), which was obtained from T. Pincus, B-clone 11 and B-clone 18 (12) from P. Jolicoeur, and B-clone E. S. from E. Scolnick. NB-tropic viruses included: WN1802BN'B-clone 1, which was cloned from WN1802BN'B, an NB-tropic virus derived from WN1802B (J. Hartley, T. Pincuis, and W. Rowe, unpublished data; see 17), which was obtained from 188
VOL. 23, 1977
RNase Ti-RESISTANT OLIGONUCLEOTIDES
189
J. Hartley. NB-18-1-clone 5 and NB-11-3-clone 1 (8, digested with 15 U of RNase T1 (Calbiochem) for 30 9) have been described. NB-E.S.-clone 744 and NB- min at 37°C. After digestion, the mixture was adE.S.-clone 745 were obtained from E. Scolnick. justed to 6 M urea with solid ultrapure urea Cells, media, and virus infections. The BALB/ (Schwarz/Mann), and a dye mixture containing 2 3T3, NIH/3T3, and Sc-i (7) cells, media, and method mg each of xylene cyanol FF and bromphenol blue of virus infections have been described (8). Chroni- per ml, 50% (wt/wt) sucrose, and 6 M urea was cally infected cells (infected at least several weeks added. The two-dimensional gel electrophoresis was before use) were used to prepare 32P-labeled virus. carried out by the method of DeWachter and Fiers To obtain B-tropic virus grown in Fv-ln cells, NIH/ (5). The first dimension was run in a 10% acrylamide 3T3 was infected with B-clone-18 at a multiplicity of gel at pH 3.5 and in the presence of 6 M urea, and infection of approximately 5. After 4 months in cul- the second dimension was run in 21.8% acrylamide ture the cells were cloned, and a cell line that pro- at pH 8.0. duced virus that gave plaques (-105 PFU/ml) on Extraction and characterization of oligonucleoBALB/3T3 but not on NIH/3T3 cells was used as a tides. After electrophoresis, the oligonucleotides source of B-tropic virus. were located by autoradiography and cut out with Preparation of 32P-labeled RNA. Confluent cul- an 8-mm-diameter cork borer. Gel disks were placed tures of chronically infected cells were incubated for in tubes containing 0.5 ml of water and 25 ,g of 4 h in phosphate-free Dulbecco-modified Eagle me- carrier RNA, and their radioactivity was quantidium containing 10% dialyzed calf serum and then tated by Cerenkov counting. After elution for 48 h at for 12 h in medium of the same composition but con- 4°C, 60 to 90% of the radioactivity could be found in taining 1 mCi of [32P]phosphate (New England Nu- the eluate. Gel disks were washed with 0.3 ml of clear Corp.) per ml. Medium was then replaced with water, and the combined eluates were lyophilized. Dulbecco-modified Eagle medium containing 20% After digestion with pancreatic RNase A (Worthingcalf serum. This medium was harvested at four 8- to ton), the oligonucleotides were analyzed as de12-h intervals, clarified by low-speed centrifugation, scribed by Adams et al. (1). and stored at -20'C. Virus was pelleted, and the Digestion with penicillium nuclease P1 (Yamasa pellets were suspended in 50 mM Tris-hydrochloride Biochemical, Tokyo) was for 1 h at 37°C in 10 mM (pH 7.5)-100 mM NaCl-1 mM EDTA (TNE buffer) Tris-hydrochloride, pH 6, at an enzyme-to-RNA and adjusted to 125 gg of carrier RNA (Sigma grade ratio (wt/wt) of 1:100. Base composition of total viral VI yeast RNA, repurified by phenol extraction) per RNA and of primary or secondary oligonucleotides ml and 1% sodium dodecyl sulfate. was done by digestion at 37°C for 30 min with a mixThis mixture was extracted two or three times ture of Takadiastase (Calbiochem; Sanzyme-R) (18) with equal volumes of a 1:1 mixture of redistilled and RNase A as described by Flanegan et al. (in phenol (buffered with TNE and chloroform-isoamyl press). The products from P1 or Takadiastase-RNase alcohol [24:1]), and the RNA was precipitated by A digestion were separated by electrophoresis on addition of 2 volumes of ethanol and storage at Whatman 3 MM paper at pH 3.5 (2). After digestion -20'C. The method of Flanegan et al. (J. B. Flane- and electrophoresis, the products were located by gan, R. F. Pettersson, V. Ambros, M. J. Hewlett, autoradiography and quantitated by cutting out the and D. Baltimore, Proc. Natl. Acad. Sci. U.S.A., in spots and counting them in a xylene-based scintilpress) was used to establish that this protocol labels lation fluid (Econofluor, New England Nuclear the individual nucleotides with equal specific activi- Corp.). ties. RESULTS To prepare 70S RNA, the ethanol precipitate Rowe and Hartley (6) have described the isowas dissolved in 10 mM Tris-hydrochloride (pH 7.5)100 mM NaCl-1 mM EDTA-0.5% sodium dodecyl lation of B-tropic virus from BALB/c mice by sulfate (SDS buffer) and centrifuged on a 15 to 30% serial passage of an extract of the spleen of nonsucrose gradient in SDS buffer in a Beckman SW41 diseased adult BALB/c mice through BALB/c rotor at 36,000 rpm for 2.5 h at 20°C. mouse embryo cells in vitro. One such isolate, 35S RNA was prepared by dissolving ethanol- designated WN1802B, has served as a prototype precipitated 70S RNA in 10 mM Tris-hydrochloride B-tropic virus for many studies and has been (pH 7.5)-i mM EDTA-0.5% SDS, heating to 100°C for 30 s, and quick-cooling on ice. This heat-dena- cloned in several laboratories. We infected tured RNA was centrifuged on a 15 to 30% sucrose BALB/3T3 cells with WN1802B and each of gradient as described above for 5 h. Also, 35S RNA three clonal isolates, B-clone 18, B-clone 11, was prepared by the method of Leis (15) with a 5 to and B-clone E.S., derived from WN1802B. Fin20% sucrose gradient containing 80% dimethyl sulf- gerprints obtained by two-dimensional gel elecoxide. RNA sequences containing polyadenylic acid trophoresis of the RNase Ti-resistant oligonuregions were selected by chromatography on poly- cleotides generated after digestion of 32P-lauridylic acid-Sephadex as described by Coffin and beled 70S RNA from each of these four viruses Billeter (4). of the Two-dimensional electrophoresis. RNA samples are shown in Fig. 1A to D. A diagram in 2. of B-clone-E.S. shown is fingerprint Fig. were adjusted to a total of 100 ug with carrier RNA and were dried on a polyethylene sheet. Digestion As expected, the fingerprints of these four Band electrophoresis were carried out essentially as tropic viruses are very similar, although two of described by Billeter et al. (3). Briefly, samples were the clones, B-clone 11 and B-clone E.S., possess
190
FALLER AND HOPKINS
J. VIROL.
one oligonucleotide that is not present in WN1802B, and B-clone E.S. also lacks one ohgonucleotide relative to WN1802B (see arrows in Fig. 1B and D). That the arbitrarily numbered oligonucleotides seen in Fig. 2 are derived from the viral genome is based on two lines of evidence. (i) The same pattern of oligonucleotides was obtained for NB-E.S.-clone 745 after digestion of either 70S RNA, 35S RNA obtained by denaturation of 70S RNA and sedimentation in 80% dimethyl sulfoxide gradients, or polyadenylic acid-selected 35S RNA (data not shown). (ii) The numbered oligonucleotides, with the exception of B-35, are present in approximately molar amounts relative to one another. Because the second dimension of the fingerprint separates oligonucleotides primarily on the basis of size, the relative molarity of the oligonucleotides can be determined by quantitating the
radioactivity present in each "spot." Further digestion of each Ti-resistant oligonucleotide with pancreatic RNase or complete digestion to mononucleotides and quantitation of the digestion products allows sizing of the Ti-resistant oligonucleotides and thus allows more accurate quantitation of their molarity. Table 1 lists the pancreatic RNase digestion products and the nucleotide length (±+ 20%) of numbered oligonucleotides of B-clone E.S. The oligonucleotide numbered B-35 in Fig. 2 appears "darker" than the two flanking oligonucleotides in the fingerprints of the B-tropic viruses (Fig. 1A-D), suggesting that this "spot" is a "doublet" consisting of two oligonucleotides that co-migrate. Quantitation of the radioactivity present in this "spot" also implies that it is present in twicemolar amount. Further analysis of spot 35 (and likewise the flanking spots 27 and 28) has been complicated by the proximity of the many unre-
10W
,qw
w
w ...-
4~~~~ ... a
0
v
4b6
--'
*
w~~~
46
0
0
0
0
B WN 1802B
A
p
9
Ar~
S
0
0
a
a
0 0~ a
NB E
I-i
WNI802BN B Clone
i-..
1.
i.-,
:". f:.
I
f, ':
F
FIG. 1. Two-dimensional gel electrophoretic fingerprints of 32P-labeled RNase Ti-resistant oligonucleotides of B- and NB-tropic viruses. B-tropic viruses: (A) WN1802B, (B) B-clone 11, (C) B-clone 18, (D) B-clone E.S. Arrows in (B) and (D) indicate oligonucleotides "unique" to B-clone 11 and B-clone E.S., respectively. Box surrounds region that is altered in NB-tropic viruses. NB-tropic viruses: Each NB-tropic virus was derived from the B-tropic virus pictured directly above it. (E) WN1802BN'B-clone 1, (F) NB-11-3-clone 1, (G) NB-18-1-clone 5, (H) NB-E.S.-clone 744, (I) NB-E.S.-clone 745. Arrows in (F and G) indicate oligonucleotides that are not present in the B-tropic virus progenitors of NB-11-3-clone 1 and NB-18-1-clone 5, respectively. The box in each figure is drawn around the region in which there is a change in all five NB-tropic viruses relative to their B-virus progenitors: one oligonucleotide is gained and one appears to be lost in this region.
VOL. 23, 1977
RNase Ti-RESISTANT OLIGONUCLEOTIDES
solved oligonucleotides migrating slightly ahead of it. Preliminary analysis of the products obtained after pancreatic RNase digestion of B-35 reveals the presence of U, C, (AG), (AAC), and several (AC)'s. Attempts to separate the B-35 doublet into its presumed component oligonucleotides, including electrophoresis on DEAE-cellulose at pH 1.9-7% formic acid, have been unsuccessful. We next infected BALB/3T3 cells with each of five NB-tropic viruses derived from the four Btropic viruses analyzed above. The origin of
191
these viruses is summarized in Fig. 3. NBtropic viruses derived from different clones of B-tropic virus or from cloned versus uncloned WN1802B must have arisen independently of one another. NB-E.S.-clone 744 and clone 745 were both derived from B-clone E.S.; however, these viruses have been shown to differ: when the virion proteins of these viruses and of Bclone E.S. are examined on SDS-polyacrylamide gels, NB-E.S.-clone 744 and clone 745 are found to possess a p30 that migrates ahead of the B virus p30 but, in addition, NB-E.S.-clone
a
0 a
0
0
S.
'a
*5
05 0aS
0
S
B
B
B-Clone 18
C
B- Clone ES
D a
9
dZ
51
4
S
0
1%
*
50
S _a
*
0 0
w
0
NB G
NB
NB-1lB--Clone 5
NB- ES- Clone 744
H 'S
SA 'S* S
%
I FIG. 1. C, D, G, H and
9 I.
NB NB-ES-Clone 745
192
FALLER AND HOPKINS
J. VIROL.
FIG. 2. Diagrammatic representation of fingerprints of B-clone E.S. and NB-E.S.-clone 744 or 745. Numbered oligonucleotides are those whose pancreatic RNase digestion products have been analyzed. In the five NB-tropic viruses, there is a new oligonucleotide, NB-34, relative to the B-tropic virus progenitors, and spot 35 is a single oligonucleotide rather than a doublee" as in the B-tropic viruses. Arrows indicate direction of migration in first and second dimensions of the gel electrophoresis. XC and B indicate position of dye markers xylene cyanol FF and bromphenol blue, respectively.
745 possesses a p15 with altered electrophoretic mobility relative to the p15 of B-clone E.S., suggesting that these two NB-tropic viruses are independent isolates (9; J. Schindler, R. Hynes, and N. Hopkins, unpublished data). Figures 1E to I show the fingerprints of the five NB-tropic viruses derived from the B viruses pictured directly above them. The NBtropic virus fingerprints look very similar to those of their B-tropic virus progenitors. This apparent similarity is further supported by the fact that (numbered) oligonucleotides that appear to migrate similarly between B-clone E.S. and NB-E.S.-clone 745 yield the same products after digestion with pancreatic RNase (Table 1).
Although the B- and NB-tropic viruses apparently share 33 to 34 of 35 to 36 large T1resistant oligonucleotides, each NB-tropic virus possesses an apparently common alteration relative to its B-tropic virus progenitor. This alteration, which is found in the region of the fingerprints enclosed by the box and is shown closeup in Fig. 4, involves the acquisition of a new oligonucleotide, designated NB-34 in Fig. 2, in each NB-tropic virus. It apparently also in-
volves the loss of one oligonucleotide from the doublet, B-35, found in each B-tropic virus, since NB-35 appears to show an intensity equal to that of the two flanking oligonucleotides. That the new oligonucleotide, NB-34, is probably the same in all five viruses is suggested by the fact that the products of pancreatic RNase digestion of NB-34 are the same for the five viruses. Analysis of oligonucleotide NB-35, like that of B-35, is hampered by contamination from the oligonucleotides migrating just above. However, the pancreatic RNase digestion products of NB-35 consist of U, C, (AG), and (AAC), and a loss of several (AC)'s relative to B-35 is apparent. The pancreatic RNase digestion products of NB-35 appear to be the same for all five NBtropic viruses. It is interesting to note the possibility of a relationship between the hypothetical oligonucleotide, which has been lost at position 35 in the NB-tropic viruses, and the new oligonucleotide, which appears in these viruses at position NB-34. NB-34 possesses several (AC's and ends in AG. Since NB-35 has lost several (AC)'s relative to B-35, it is conceivable that one oligonucleotide in B-35 has been al-
VOL. 23, 1977
RNase Ti-RESISTANT OLIGONUCLEOTIDES
TABLE 1. RNase Ti-resistant oligonucleotides of Bclone E.S. and its NB-tropic derivatives Oligonucleotide
Chain length (nucleotides)c
Composition"
no. a
1
5U, 8C, 4(AC), 2(AU).
2
2(A2C), G 10U, 15C, (AU), (A3C), (A4C), G 2U, 7C, (AU),
3
\2C) -.,
(AXC), (AXC),
32
+-
20%
40
(A2G)
4
5
6 7 8 9 110 111 112
1U, 5C, (AC), (AU), (A2U), (A5C) G 1U, 6C, (AC), 2(AU), (A2U), (A4C) G 2U, 5C, 2(A2C), (A2U), (AG) 5U, 10C, 2(AC), G 4U, 7C, 2(AC), 3(AU), (AG) 6U, 5C, 3(AC), (AU), (A2C), G 2U, 13C, 3(AC), (A3C), G 3U, 12C, 2(AC), (A3C), (AG) 1U, 4C, 3(AC), (A2U), (A4G) 2U, 8C, 2(AC), (A2C), G 1U, 2C, 2(A2C), (A5C), (AG) 5U, 6C, 2(A2U), (A5U), G 7U, 8C, 2(AC), (AU), G 8U, llC, G 4U, 3C, (AC), 2(AU), (A4G) 4U, 5C, (AC), (AU), (A2U), (AG) 4U, 6C, 2(AC), 2(AU), G 3U, 3C, 3(AU), (A2C), G 5U, 2C, 2(AC), (A2U), G 6U, 3C, (AC), (A2G) G 5U, 4C, (AC), 3(AU) G 4U, 3C, (AU), (A2U) G 5U, 1C, 2(AU), (AG)
193
tered to generate NB-34. However, since oligonucleotides with similar composition will have similar electrophoretic mobilities in the first dimension, the fact that NB-34 and the "postulated" oligonucleotide apparently possess common pancreatic RNase digestion products does not necessarily imply a relationship between these two oligonucleotides. In addition to the two common changes, two of the five NB-tropic viruses, NB-11-3-clone 1 and NB-18-1-clone 5, each possess a new, "unique" oligonucleotide relative to their B-virus progenitors (see arrows, Fig. 1F and G). We have examined the fingerprints of Bclone 18, B-clone E.S., NB-18-1-clone 5, and NB-E.S.-clone 744 viruses grown in Sc-1 cells (a cell line that does not show Fv-1 restriction), NB-11-3-clone 1 grown in NIH/3T3 cells, and Bclone 18, which had grown in NIH/3T3 cells but retained its B-tropism. The fingerprints showed the same pattern of oligonucleotides as those of the corresponding viruses grown in BALB/3T3 cells.
DISCUSSION Fingerprints of the RNase Ti-resistant oligonucleotides of a B-tropic virus of BALB/c and five of its NB-tropic derivatives were shown to .5 24 be similar. Approximately 33 of 35 large very 16 22 Ti-resistant oligonucleotides appeared to be L7 20 shared by these viruses. However, all five NBL8 18 tropic viruses were found to possess a common L9 18 alteration relative to their B-virus progenitor. This alteration involves the acquisition of one 10 19 oligonucleotide and, probably, the loss of one 211 16 oligonucleotide. Preliminary results are not in212 15 consistent with the possibility that the new 213 15 "NB-oligonucleotide" could have been gener214 18 ated by an alteration in the oligonucleotide that 2 S5 13 has disappeared relative to the B-tropic virus; 216 12 2U, 8C, 2(AC), (A2C), however, the data on this point are far from 19 19 2 (AG) conclusive. Any number of mechanisms-sin10 2U, 9C, 3(AC), (AG) 19 gle base change, deletion, insertion, or recom3 11 3U, 5C, 3(AC), (A5C), G 21 bination with a different endogenous virus3 12 1U, 7C, (AC), (A2C), 18 could generate the alterations in the RNase T13 (A3C), G resistant oligonucleotides that we have ob13 8U, 1C, (AC), (A2U), G 15 served in the NB-tropic viruses. NB-314 2U, 2C, 4(AC), (A2U), 17 A question of obvious interest is whether (AG) these alterations reflect a change(s) that is rea Numbers refer to oligonucleotide spots diasponsible for the change from B- to NB-tropism. gramed in Fig. 2. If homologous spots of B-clone We cannot answer this question at present. The E.S. and its NB-tropic derivatives have the same facts that this change occurred in five viruses composition, they are listed only once. The oligonu- isolated in three different laboratories, that the cleotide that is present in NB-E.S.-clones 744 and fingerprints appear to be unaltered when the 745 but not in B-clone E.S. is designated NB-34. b Compositions are determined by quantitation of NB-tropic viruses are grown in NIH/3T3, 113 114
18 17
the products of pancreatic RNase digestion. Average values for at least four determinations are given and are rounded to the nearest integer.
c Chain length was computed by addition of the nearest integers of the molar yields.
194
J. VIROL.
FALLER AND HOPKINS WN1802B B-clone 11
B-clone 18
WN1802BN'B-clone 1
B-clone E.S.
NB-E.S.-clone 744
NB-18-1-clone 5
NB-E.S.-clone 745
NB-11-3-clone 1
FIG. 3. Origin of B- and NB-tropic viruses. B-tropic viruses: WN1802B (6) is an uncloned B-tropic virus derived by serial passage of the extract of a spleen of a non-diseased adult BALBIc mouse through secondary BALBIc embryo cells; B-clone 18 (12), B-clone 11 (12), and B-clone E.S. (E. Scolnick, unpublished data) are clonal isolates derived from WN1802B. NB-tropic viruses: WN1802BN'B-clone 1 is a clonal isolate derived from WN1802BN'B (J. Hartley, T. Pincus, W. Rowe, unpublished data) derived from WN1802B by serial passage on secondary NIH Swiss embryo cells; NB-18-1-clone 5 (9), NB-11-3-clone 1 (9), NB-E.S.-clone 744 (E. Scolnick, unpublished data) and NB-E.S.-clone 745 (E. Scolnick, unpublished data) are clonal isolates derived from B-clone 18, B-clone 11, and B-clone E.S., respectively, by serial passage through NIHI3T3 cells.
: Do
*
B
_NB
FIG. 4. Close-up of the region of the fingerprints in which NB-tropic viruses differ from their B-tropic virus progenitors. (B) Close-up of region surrounded by a box in Fig. IA (B-tropic virus WN1802B). (NB) Close-up of this region from Fig. lE (NB-tropic virus WN1802BN'B-clone 1).
BALB/3T3, or Sc-1 cells, and that a B-tropic virus grown in NIH/3T3 cells, which retained its B-tropism, did not show the alterations observed in the NB-tropic viruses are at least consistent with the possibility that the changes observed may be related to the genetically stable change to NB-tropism. We are currently attempting to "map" the new oligonucleotide present in the NB-tropic viruses relative to the 3' end of the genome (4, 22). Although the gene order of murine leukemia viruses is not yet known, it seems quite likely that it will be similar to that of the avian type C viruses. Furthermore, we are currently analyzing the RNase Ti-resistant oligonucleotides of an N-tropic virus of BALB/c and 21 recombinants between this virus and the Btropic virus, B-clone 18, described here (8). Since these recombinants appear to possess different combinations of N- and B-virus virion proteins (10; J. Schindler, R. Hynes, and N. Hopkins, unpublished data), this analysis may
yield a map order for these proteins. Hopkins et al. (9) have found that the NB-tropic viruses analyzed here possess an altered p30 relative to their B-tropic virus progenitors. It will be interesting to see whether the new NB-oligonucleotide lies in a region of the genome where the gag gene, which codes for p30 and other internal virion proteins, is expected to reside. ACKNOWLEDGMENTS We thank John Coffin for a first-hand demonstration of this method of RNA fingerprinting and for very helpful discussions. We thank John Schindler for analyzing the proteins of NB-E.S.-clone 744 and 745 viruses. We thank Paul Gottlieb and Phillip Sharp for helpful criticisms. This work was supported by Public Health Service research grants from the National Cancer Institute (CA 19308 to N. H., and CA 14051 to the Center for Cancer Research, Massachusetts Institute of Technology).
ADDENDUM IN PROOF Preliminary determinations of the relative order of the large Ti-resistant oligonucleotides of NB-ESclone 745 with respect to the 3' end of the genome
VOL. 23, 1977
RNase Ti-RESISTANT OLIGONUCLEOTIDES
indicate that oligonucleotide NB-34 is among the four oligonucleotides closest to the 5' end. LITERATURE CITED 1. Adams, J. M., P. G. N. Jeppersen, F. Sanger, and B. G. Barrell. 1969. Nucleotide sequence from the coat protein cistron of R17 bacteriophage RNA. Nature (London) 223:1009-1014. 2. Barrell, B. G. 1971. Fractionation and sequence analysis of radioactive nucleotides, p. 751-758. In G. L. Cantoni and D. R. Davies (ed.), Procedures in nucleic acid research, vol. II. Harper and Row, New York. 3. Billeter, M. A., J. T. Parsons, and J. M. Coffin. 1974. The nucleotide sequence complexity of avian tumor virus RNA. Proc. Natl. Acad. Sci. U.S.A. 71:35603564. 4. Coffin, J. M., and M. A. Billeter. 1976. A physical map of the Rous sarcoma virus genome. J. Mol. Biol. 100:293-318. 5. DeWachter, R., and W. Fiers. 1972. Preparative twodimensional polyacrylamide gel electrophoresis of 32P-labeled RNA. Anal. Biochem. 49:184-197. 6. Hartley, J. W., and W. P. Rowe. 1975. Host-range restriction of murine leukemia viruses in mouse embryo cell cultures. J. Virol. 5:221-225. 7. Hartley, J. W., and W. P. Rowe. 1975. Clonal cell lines from a feral mouse embryo which lack -host-range restrictions for murine leukemia viruses. Virology 65:128-134. 8. Hopkins, N., P. Traktman, and K. Whalen. 1976. Ntropic variants obtained after co-infection with Nand B-tropic murine leukemia viruses. J. Virol. 18:324-331. 9. Hopkins, N., J. Schindler, and R. Hynes. 1977. Six NBtropic viruses derived from a B-tropic virus of BALB/c have altered p30. J. Virol. 21:309-318. 10. Hopkins, N., J. Schindler, and P. Gottlieb. 1977. Evidence for recombination between N- and B-tropic murine leukemia viruses. J. Virol. 21:1074-1078. 11. Huang, A., P. Besmer, L. Chu, and D. Baltimore. 1973. Growth of pseudotypes of vesicular stomatitis virus with N-tropic murine leukemia virus coats in cells resistant to N-tropic viruses. J. Virol. 12:659-662.
195
12. Jolicoeur, P., and D. Baltimore. 1975. Effect of the Fv-1 locus on the titration of murine leukemia viruses. J. Virol. 16:1593-1598. 13. Jolicoeur, P., and D. Baltimore. 1976. Effect of Fv-1 gene product on proviral DNA formation and integration in cells infected with murine leukemia viruses. Proc. Natl. Acad. Sci. U.S.A. 73:2236-2240. 14. Krontiris, T. G., R. Soeiro, and B. N. Fields. 1973. Host restriction of Friend leukemia virus. Role of the viral outer coat. Proc. NatI. Acad. Sci. U.S.A. 70:25492553. 15. Leis, J. P. 1976. RNA-dependent DNA polymerase activity of RNA tumor virus. VI. Processing mode of action of avian myeloblastosis virus polymerase. J. Virol. 19:932-939. 16. Lilly, F. 1967. Susceptibility of two strains of Friend leukemia virus in mice. Science 155:461-462. 17. Lilly, F., and T. Pincus. 1973. Genetic control of murine viral leukemogenesis. Adv. Cancer Res. 17:231277. 18. Niramaru, M., T. Uchida, and F. Egami. 1966. Ribonuclease preparation for the base analysis of polyribonucleotides. Anal. Biochem. 17:135-142. 19. Pincus, T., J. W. Hartley, and W. P. Rowe. 1971. A major genetic locus affecting resistance to infection with murine leukemia viruses. I. Tissue culture studies of naturally occurring viruses. J. Exp. Med. 133:1219-1233. 20. Rein, A., S. V. S. Kashmiri, R. H. Bassin, B. I. Gerwin, and G. Duran-Troise. 1976. Phenotypic mixing between N- and B-tropic murine leukemia viruses: infectious particles with dual sensitivity to Fv-1 restriction. Cell 7:373-379. 21. Sveda, M. M., and R. Soeiro. 1976. Host restriction of Friend leukemia virus: synthesis and integration of the provirus. Proc. Natl. Acad. Sci. U.S.A. 73:23562360. 22. Wang, L., D. Galehouse, P. P. Mellon, P. Duesberg, W. S. Mason, and P. K. Vogt. 1976. Mapping oligonucleotides of Rous sarcoma virus RNA that segregate with polymerase and group-specific antigen markers in recombinants. Proc. NatI. Acad. Sci. U.S.A. 73:3952-3956.
