Radiation leukemia virus contains two distinct viral RNAs

Cell, Vol. 19, 643-652, March 1980, Copyright © 1980 by MIT

Radiation Leukemia Virus Contains Two Distinct Viral RNAs

Simone ManteuiI-BruUag, Sai-ling Liu and Henry S. Kaplan Cancer Biology Research Laboratory Department of Radiology Stanford Medical School Stanford, California 94305

Summary We have analyzed the RNA genome of RadLV/VLz, a highly oncogenic murine leukemia virus. This virus is produced by a permanent cell line derived from a radiation leukemia virus-induced thymic lymphoma of C 5 7 B L / K a mice. Two distinct RNA components were found in the virions: a 70S dimer containing two 8 kb RNA subunits and a 54S dimer containing two 5.6 kb RNAs. A nononcogenic retrovirus, B L / Ka(B), endogenous in the same strain of mice, contains only 8 kb viral RNA subunits. The linkages between both RadLV/VLz dimers have identical thermal stabilities. Both dimers can serve as primer templates for reverse transcriptase and both produce very similar "strong-stop" cDNAs 147 _ 1 bases long. Sequences at the 5' end of the 5.6 kb subunit contain the genes for the viral proteins p15 and p12, but the gene for p30 is either absent or partially deleted. In vitro translation of the 5.6 kb RNA yields a 100,000 molecular weight protein containing antigenic determinants which react with antibody to p15 but not with antibody to p3O. In addition, cells producing RadLV/VL3 virus synthesize a novel 1.6 kb poly(A)-containing cytoplasmic RNA which shows very little if any homology with BL/Ka(B) viral sequences. Introduction We have analyzed the genomic composition of the radiation leukemia virus (RadLV). The biology of RadLV is particularly well characterized. This virus can be consistently isolated from lymphoid tumors of the thymus which develop after the controlled X-irradiation of C57BL/Ka mice (Kaplan and Brown, 1952; Lieberman and Kaplan, 1959; Kaplan, 1967). The essential role of the thymus in the development of leukemia following irradiation has been extensively documented (Kaplan, 1950; Carnes et al., 1956). RadLV is a highly oncogenic virus capable of inducing neoplastic transformation in susceptible target cells in the thymus, bone marrow, spleen and fetal liver (Kaplan and Lieberman, 1976; Lieberman and Kaplan, 1976). The predilection of RadLV for cells of T lymphocyte lineage is remarkable, and not yet understood; it contrasts markedly with that of another murine leukemia virus (MuLV), Abelson murine leukemia virus, which transforms cells of the B lymphocyte

lineage and will not transform T lymphocytes (Abelson and Rabstein, 1970; Sklar, White and Rowe, 1974; Rosenberg and Baltimore, 1976; Boss, Greaves and Teich, 1979). To date, the molecular biologies of these two viruses have not been compared; the mechanism of RadLV oncogenicity is unknown and the transforming product(s) coded for by the RadLV genome remains unidentified. We wished to characterize the putative oncogenic sequences of RadLV and to determine their organization within the viral genome. We have compared the genomes of RadLV/VL3, a virus produced in high titer by BL/VL3, an established cell line derived from a RadLV-induced thymic iymphoma (Decleve et al., 1978; Lieberman et al., 1979), and BL/Ka(B), an endogenous virus of C57BL/Ka mice which, unlike RadLV, replicates well on fibroblasts but not in the thymus and is not oncogenic (Decleve et al., 1976, 1978). We now report that RadLV/VL3 viral RNA contains two distinct subunits 8 and 5.6 kb in length, whereas the only viral RNA detected in BL/Ka(B) is an 8 kb RNA subunit. We show that the two RNA subunits of RadLV/VL3 share at least 0.9 kb of sequences at their 5' ends and that the 5.6 kb subunit can code in vitro for the synthesis of a 100,000 molecular weight protein which carries antigenic determinants to viral internal structural proteins. We have also identified the presence of two subgenomic viral RNAs in the cytoplasm of cells producing RadLV/ VLs virus. Results RadLV/VLz Contains Two RNA Dimers with Different Sized Subunits RadLV/VL3 viral preparations contain several RNAs of various molecular weights when analyzed on agarose gels under denaturing conditions (Figure 1, lane 1). The predominant species present were identified as host ribosomal RNAs by their size (4.4 and 1.9 kb) and by the fact that they do not contain poly(A) (data not shown). When the virus was labeled with 3Huridine for 4.5 hr, the majority of the radioactivity was recovered in two higher molecular weight RNAs (8 +_ 0.3 kb and 5.6 + 0.2 kb; Figure 1, lane 3). These two RNA species could also be detected visually by staining, and appeared in equimolar amounts (Figure 1, lane 1). More than 80% of these larger RNAs bound to oligo(dT)-cellulose, and hence contained poly(A) (data not shown). When the RNAs from the nononcogenic virus BL/Ka(B) were analyzed by the same procedure, unlabeled host rRNAs were also present (Figure 1, lanes 2 and 5). This virus, however, contained only a single RNA species larger than the rRNAs. This larger RNA was indistinguishable in size from the 8 kb RNA present in RadLV/VL3 virus (8.0 + 0.3 kb), and the majority of the radioactive label

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Figure 1. DeterminationoftheMolecularWeightsofRadLV/VL3RNA and BL/Ka(B) RNA on Denaturing Gels RadLV/VL3 and BL/Ka(B) viruses were labeled with 3H-uridine and purified, and the RNA was extracted in the presence of yeast RNA carrier as described in Experimental Procedures. The RNAs were dissolved in half-strength electrophoresis buffer containing 10 mM methylmercury hydroxide (Alfa Ventron Products) and analyzed on 1.2% agarose slab gels also containing 10 mM methylmercury hydroxide, Electrophoresis was for 16 hr at 1.3 V/cm, Samples 1, 2 and 5, 6 were analyzed independently. (Lane 1) RadLV/VL3 RNAs from 5 X 10 8 cells (3 x 10 5 cpm); (lanes 2 and 5) BL/Ka(B) RNAs from 1.5 x 10 s cells (4 x 10 s cpm); (lanes 3 and 4) fluorograms of 1 and 2, respectively (2 days exposure); (lane 6) 0.5 /~g of total cytoplasmic RNA from SC-1 cells.

was present in this species (Figure 1, lane 4). No 5.6 kb RNA was detected either on the stained gel or in the fluorograms. The 8 kb RNA from BL/Ka(B) virus also contained poly(A). Since the RNA genomes of several retroviruses have been shown to be dimers of two RNA molecules (Bender and Davidson, 1976; Bender et al., 1978), we decided to examine the sedimentation properties of the RadLV/VL3 8 kb and 5.6 kb RNAs to determine whether either or both of them existed in a more complex form in the virion. Viral RNA was resuspended in buffered solutions of 8 M urea, heated at 40°C and then sedimented in glycerol gradients. These conditions are known to dissociate RNA aggregates while preserving the structural integrity of high molecular weight RNA (Kourilsky et al., 1970; Manteuil, 1973), thus permitting the resolution of RNAs on velocity gradients. When the RadLV/VL3 RNA was heated at 40°C in the presence of 0.3 M NaCI and 8 M urea, two major RNA species were resolved sedimenting at 70S and 54S (Figure 2A). A minor peak was reproducibly found at 31 S. When salt was omitted from the 8 M urea preincubation mixture, the RNAs sedimented much more slowly after the heat treatment (Figure 2B). The sedimentation velocities of these

Figure 2. RadLV/VL3 RNAs Sediment As Dimers 3H-RadLV/VL3 RNAs (~5000 cpm) were analyzed on glycerol velocity gradients as described in Experimental Procedures. The RNAs were preincubated at 40°C for 5 rain in solutions containing 8 M urea and 0.3 M NaCI (A) or with no NaCI (B) prior to sedimentation. The gradients were run for 3 hr at 22°C, 39K rpm in a Beckman SW40 rotor. Mouse ribosomal RNAs labeled with 14C-uridine were added as internal markers. After centrifugation, the gradients were fractionated and the total TCA-precipitable ~H and ~4C radioactivity was measured.

species (38S and 31 S) are those expected for individual RNA molecules 8 kb and 5.6 kb long. By analogy with other viruses (Manuel, Delius and Duesberg, 1974; Beemon et al., 1976; Bender et al., 1978; Maisel et al., 1978), this analysis allows us to conclude that both high molecular weight RadLV/VL3 RNAs are present in the virion in forms which sediment as dimers. The minor species sedimenting at 31S in Figure 2A may be a 5.6 kb viral RNA which has not yet matured into its dimeric configuration (Canaani, Helm and Duesberg, 1973), but this possibility was not tested. We next compared the stability of the linkage associating each RNA dimer by heating them in solutions of decreasing ionic strength. The 70S and 54S species were first separated from each other on two successive glycerol gradients like that shown in Figure 2A. Dimers prepared in this way were at least 90% free of cross-contamination (Figures 3A and 3F). These separated RNA dimers were then incubated at 40°C at decreasing salt concentrations ranging from 0.35 M NaCI to no salt. We were surprised to find that after preincubation in 0.14 M NaCI all of the 70S RNAs now sedimented at 56S and that the 54S RNAs sedimented at 48S (Figures 3B and 3G). This large shift in sedimentation is probably due to an unfolding of the complex structure and not a decrease in molecular weight, because the change in sedimentation is partially reversible when the salt is returned to 0.35 M

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NaCI for a n o t h e r 5 min at 4 0 ° C ( F i g u r e s 3C and 3H). S u c h d r a m a t i c c h a n g e s in s e d i m e n t a t i o n p r o p e r t i e s h a v e b e e n d e s c r i b e d for the u n f o l d i n g of the c o m p a c t S V 4 0 m i n i c h r o m o s o m e ( 7 0 S ) to t h e o p e n b e a d e d s t r u c t u r e ( 5 5 S ) w h i c h is also f a v o r e d at l o w ionic s t r e n g t h s ( C h r i s t i a n s e n and Griffith, 1 9 7 7 ) . By a n a l o g y , it is p o s s i b l e that the c o n v e r s i o n of the 7 0 S and 5 4 S R N A c o m p l e x e s of R a d L V / V L 3 to 5 6 S and 4 8 S , r e s p e c t i v e l y , m a y c o r r e s p o n d to a d r a s t i c c h a n g e in their conformation which nevertheless preserves their d i m e r l i n k a g e s t r u c t u r e . F u r t h e r m o r e , t h e s e 5 6 S and 4 8 S s p e c i e s c o u l d be d i s s o c i a t e d into the 3 8 S and 31 S m o n o m e r s at e v e n l o w e r ionic s t r e n g t h s (Figures 3D and 3E, and 31 and 3J). T h e l i n k a g e s h o l d i n g the t w o d i f f e r e n t d i m e r s tog e t h e r h a v e identical stabilities. F i g u r e 3 K d e s c r i b e s the " m e l t i n g " profile of the 7 0 S a n d 5 4 S R a d L V / V L 3 R N A s as a f u n c t i o n of ionic s t r e n g t h . A 5 0 % c o n v e r sion to t h e m o n o m e r i c c o n f o r m a t i o n o c c u r s in 0.1 1 2 M Na ÷ and 8 M u r e a at 4 0 ° C (or at 3 9 ° C in 0.1 M Na+; M a n d e l and M a r m u r , 1 9 6 8 ) for both R N A c o m p l e x e s . A s s u m i n g that the p r e s e n c e of 8 M u r e a l o w e r s the stability of t h e s e l i n k a g e s b y 2 2 ° C , as it is k n o w n to d o for n o r m a l R N A s e c o n d a r y s t r u c t u r e s ( K o u r i l s k y et al., 1 9 7 0 ) , the e x p e r i m e n t d e s c r i b e d in F i g u r e 3 s u g g e s t s that the l i n k a g e s w o u l d melt at 61 °C in 0.1 M NaCI. This v a l u e is in p e r f e c t a g r e e m e n t with that g i v e n b y B e n d e r et al. ( 1 9 7 8 ) for the "Tm" of d i m e r s of the F r i e n d m u r i n e virus c o m p l e x and the M o l o n e y m u r i n e l e u k e m i a a n d s a r c o m a viruses. While the e x a c t p h y s i c a l n a t u r e of this l i n k a g e s t r u c t u r e is still not k n o w n , t h e s e d a t a i n d i c a t e that it has the s a m e sensitivity to the d e n a t u r i n g a g e n t u r e a as d o helical R N A s t r u c t u r e s (see m o d e l p r o p o s e d b y Haseltine, M a x a m a n d Gilbert, 1 9 7 7 ) . RadLV/VLs

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Figure 3, Dissociation of RadLV/VL370S and 54S RNA Dimers into 38S and 31S Monomers at Low Ionic Strength Purified 70S and 54S RNA dimers were preincubated at 40°C in 10 mM Tris-HCI (pH 7.4), 1 mM EDTA, 8 M urea containing decreasing NaCI concentrations as indicated in each panel and then analyzed on glycerol velocity gradients as in Figure 2. The dissociation of the 70S dimer is shown in (A), (B), (D) and (E); that of the 54S dimer is shown in (F), (G), (I) and (J). (C) and (H) show the sedimentation patterns of RNA molecules which were first preincubated at 40°C in the 0.14 M NaCI concentration for 5 rain, followed by an increase in the NaCI to

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We c o m p a r e d the 5' t e r m i n a l s e q u e n c e s of the 7 0 S and 5 4 S d i m e r s from R a d L V / V L 3 R N A by using t h e m as t e m p l a t e s for a v i a n m y e l o b l a s t o s i s virus ( A M V ) r e v e r s e t r a n s c r i p t a s e . This e n z y m e is k n o w n to utilize the t R N A pr° w h i c h is a s s o c i a t e d with the m u r i n e retrovirus g e n o m e s as a p r i m e r for s y n t h e s i s of a DNA s e q u e n c e c o m p l e m e n t a r y to the 5 ' t e r m i n u s of the viral t e m p l a t e (Coffin et al., 1 9 7 8 ; P e t e r s and Dahlberg, 1 9 7 9 ) . F i g u r e 4 s h o w s t h a t the m a j o r p r o d u c t of such r e a c t i o n s w a s a c D N A 1 4 7 +_ 1 b a s e s long for b o t h t e m p l a t e s . T h e p r i m a r y p r o d u c t of this in vitro r e a c t i o n is r e f e r r e d to as s t r o n g - s t o p DNA (ssDNA;

0.35 M and another 5 min incubation at 40°C. 14Cqabeled mouse ribosomal RNAs were added to each gradient as an internal marker (arrows). Gradients were fractionated and precipitated as in Figure 2. The area under each peak was integrated and the percentage of monomers (38S and 31S) were plotted against the molarity of NaCI (K). The sedimentation analysis of dimer molecules incubated in the presence of 0.05 M, 0.1 M, 0.114 M, 0.16 M and 0.2 M NaCI are not shown in this figure.

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Haseltine et al., 1976). Not only were the lengths of the ssDNA identical for both RNAs, but the patterns of DNA fragments smaller than 147 bases were also indistinguishable. These DNA fragments correspond to secondary structures or sequences within the RNA template at which the reverse transcriptase stops preferentially (Hasettine et al., 1976). From the above observations, and from our finding that strong-stop cDNAs made using either 70S or 54S RadLV/VLs RNA templates hybridize to the same extent to the 8 kb monomers (data not shown), we conclude that the 5' termini of the 8 and 5.6 kb monomers have very similar sequences.

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Figure 4. Comparisonof cDNAs Synthesized at the 5' Ends of the Purified 7OSand 54S RadLV/VL~RNA TemplatesUsingthe Naturally Associated tRNA Primers Approximately0.1 /~g of purified RNA dimers were transcribed into cDNA in the presence of 6/~M e-32P-dCTP and 1 mM of the other cold deoxynucleosidetriphosphates, as described in Experimental Procedures. RadLV/VL3 54S RNA (A) and (D) and 70S RNA (B) and (E) were used as templates.Both (A) and (D) and (B) and (E) are pairs of autoradiogramsof the same sample at two different exposures

Putative Viral RNA Messages Are Present in Cells Producing RadLV/VLs Several investigators have detected two species of viral RNA, tentatively identified as messenger RNAs, in cells producing retroviruses whose genome organization is 5'-gag-pol-env-3' (Fan and Baltimore, 1973; Hayward, 1977; Weiss, Varmus and Bishop, 1977). The length of the larger RNA species is indistinguishable from that of the genomic RNA (35S), and it is probably utilized for the translation of the gag and pol proteins (Philipson et al., 1978). Another viral RNA, 21 S in size, contains a short sequence from the 5' end of the 35S RNA spliced onto the 3' third of the genome, and this RNA probably encodes the env protein p r o d u c t (Rothenberg, Donoghue and Baltimore, 1978). When poly(A)-containing cytoplasmic RNA from BL/VL3 cells was analyzed for the presence of viral-specific sequences, four RNA species, having lengths of 8.0 kb, 5.6 kb, 3.4 kb and 1.6 kb, were detected. The largest of these (8.0 kb), though not discernible as a discrete band in the experiment illustrated in Figure 5 due to degradation, was the most abundant viral RNA species observed in other experiments of similar design (data not shown). By analogy with the other retroviruses described above, the 8 kb and 5.6 kb RNAs are probably very similar to the t w o RNAs found in RadLV/VLs virions, with the larger coding for the precursors of the gag and pol proteins and the smaller having an as yet undetermined function. Hybridization of a B L / K a ( B ) - s p e c i f i c probe to these putative messengers revealed that a randomly primed cDNA from the B L / K a ( B ) g e n o m e did not hybridize detectably with the 1.6 kb RNA sequences (Figure 5B). These experiments suggest the presence of two s u b g e n o m i c viral m R N A s in cells producing R a d L V / V L s virus, whose relationship to the viral RNAs has yet to be determined. which show either the "strong-stop" DNA (A) and (B) or shorter structural pauses (D) and (E) in the reverse-transcription reaction. The molecular weight markers are two sequence analyses in either direction from the Hinf site within the repeating subunit of the 1,688 satellite DNA of Drosophila melanogaster (Hsieh and Brutlag, 1979). The fragments shown result from partial cleavage at G residues according to the method of Maxam and Gilbert (1977).

The Genomeof RadLV/VL3 647

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Figure 5. ViralRNAsin BL/VL3Cells Poly(A)-containingRNAswereisolatedfrom 0.5 mg of totalcytoplasmic BL/VL3 RNA, denaturedin glyoxal,fractionatedon an agarose gel,transferredto DBMpaperand hybridizedto variouscDNAprobes labeledusingc~-32P-dCTPas describedin ExperimentalProcedures. The probesusedwere(A) a randomlyprimedcDNAmadeon RadLV/ VL35.6 kb RNA;(B) a randomlyprimedcDNAmadeon BL/Ka(B)8 kb RNA. Due to degradation,no discreteband is seen at 8.0 kb in this experiment.However,this viral RNA specieswas readilyidentifiable in othersuch experiments.

Gag-Specific Sequences Are Present in the 5.6 kb RadLV/VL3 RNA Figure 6 shows the in vitro translation products from both the 8 kb and the 5.6 kb RadLV/VL3 RNAs. With either template, two major protein products were identified. The 8 kb RNA produced 63,000 and 70,000 molecular weight proteins which are similar in size to those produced by in vitro translation of Moloney MuLV RNA (Mo-MuLV; Kerr et al., 1976; Edwards and Fan, 1979). The in vitro translation products of the 8 kb RNA from BL/Ka(B) were also two proteins of 63,000 and 70,000 molecular weights (data not shown), which were synthesized in a ratio similar to those from the RadLV/VL3 8 kb RNA (Figure 6B). In the case of Mo-MuLV, the smaller protein is the precursor of the internal structural proteins (Pr65~ag), while the larger protein becomes glycosylated and appears at the surface of infected cells (Ledbetter, Nowinski and Eisenman, 1976; Edwards and Fan, 1979). The two major products of translation of the 5.6 kb RNA were 30,000 and 36,000 molecular weight proteins (Figure 6C). The small amounts of the 30,000 and 36,000 molecular weight proteins in Figures 6B and 6E are ascribed to residual (<10%) cross-contamination of the 8.0 kb viral RNA species

by the 5.6 kb species (Figure 3A). Consistent with that interpretation is the fact that we could not detect the 30,000 and 36,000 molecular weight proteins among the in vitro translation products of the 8 kb RNA from the BL/Ka(B) virus (data not shown). To determine the nature of these translation products, we have characterized their reactivity with a series of antisera (provided by M. Strand and J. T. August). We used antisera prepared against either the Rauscher MuLV gag proteins (Strand and August, 1976), whose gene order is NH2-pl 5-pl 2-p30-pl0COOH (Murphy and Arlinghaus, 1978), or against the Gross MuLV virus gag proteins, whose gene order is NH2-p12-p14-p30-p10-COOH (M. Strand, personal communication). The Rauscher p15 and Gross p12 and the Rauscher pl 2 and Gross p14 are functionally identical protein pairs. For simplicity we have adopted the Rauscher MuLV gag protein terminology, since the gene order of RadLV/VL3 gag proteins is not known. Figures 6E and 6F show that all protein products of in vitro translation of the 8 and 5.6 kb RNAs were precipitable by a combination of antibodies directed against the p15 and p12 proteins (cx-p15 + ~-p12). To our surprise, a 100,000 molecular weight protein which was a minor translation product of the 5.6 kb RNA (Figure 6C) was a major constituent of the products found after precipitation with the c~-pl 5 + ~-pl 2 antisera (Figure 6F). None of the translation products from the 5.6 kb RNA contained p30 antigenic determinants (Figure 6H). That the 100,000 molecular weight protein was indeed specifically a protein product of the 5.6 kb RNA was indirectly confirmed by its absence from the proteins translated from the 8 kb RNA (which contains the p30 gene) after their precipitation with ~x-p30 antibodies (Figure 6G). The antisera directed at pl 2 alone (~x-pl 2) precipitated the 30,000 and 36,000 molecular weight peptides but not the 100,000 molecular weight protein (Figure 6J). It is possible, however, that pl 2 determinants are present in the 100,000 molecular weight protein but are masked. Such a phenomenon has been described for other viral proteins (Ledbetter et al., 1978). Likewise, the 70,000 molecular weight protein, which in other viruses has been shown to contain pl 2 determinants, is only poorly precipitated with ~x-pl 2 compared to the precursor Pr63 gag (Figure 61). From these data we conclude that the 5.6 kb RadLV/VL3 RNA contains p15 and p12 gene sequences but few or no p30 sequences, and that it can code for the synthesis of a 100,000 molecular weight polyprotein containing gag antigenic determinants.

Discussion We have shown that RadLV/VL3, a highly oncogenic virus produced by a cell line derived from a RadLVinduced thymic lymphoma, contains two distinct viral

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Figure 6. In Vitro Translation of RadLV/VL3 Viral RNAs Purified 70S (~0.4/~g) and 54S (-0.2/Lg) RadLV/VL3 RNAs were denatured and translated in a nuclease-treated rabbit reticulocyte lyeate system containing 35 S-methionine. Translation products were separated by electrophoresis on gradient acrylamide gels [16 cm long in (A), (B), (C), (D), (E), (F), (G), (H), and (G); 9 cm long in (I) and (J)] and visualized by fluorography. The translation products are from (A) no added RNA; (B) 8 kb RNA; (C) 5.6 kb RNA; (E) and (F) as in (B) and (C) but precipitated with ~x-pl5 Rauscher + ~x-pl2 Gross + ~x-p14Gross antisera; (G) and (H) as in (B) and (C) but precipitated with ~x-p30 Rauscher +~x-p30 Gross antisera; (I) and (J) as in (B) and (C) but precipitated with ~x-pl4 Gross. P14 from the Gross virus is the internal core phosphoprotein. To avoid confusion, the Rausher terminology for the gag gene products was used (see text). Molecular weight standards were provided by the/7- B' subunits of the E. coil RNA polymerase (165,000 and 155,000); human transferrin (75,000); bovine serum albumin (68,000); creatine kinase (39,000); and cytochrome C (13,000). R N A s 8 and 5.6 k b in length w h i c h are both p r e s e n t in t h e virions in t h e form of c o m p l e x e s w h i c h s e d i m e n t as h o m o d i m e r s . W e have d e m o n s t r a t e d that the t w o R N A s have v e r y similar 5' terminal s e q u e n c e s which i n c l u d e a 147 +_ 1 base s t r o n g - s t o p s e q u e n c e and c o d i n g r e g i o n s for the viral p r o t e i n s p l 5 and p l 2. The c o d i n g region for p 3 0 is either a b s e n t or partially d e l e t e d in the 5 . 6 k b RNA. We have also d i s c o v e r e d t w o novel viral R N A s in cells p r o d u c i n g R a d L V / V L 3 . As in cells p r o d u c i n g o t h e r t y p e s of murine l e u k e m i a viruses c o n t a i n i n g e x c l u s i v e l y an 8 kb g e n o m e , we

find an 8 k b p o l y ( A ) - c o n t a i n i n g R N A and a 3.4 k b R N A ( R o t h e n b e r g et al., 1978). In a d d i t i o n to these, w e also find a 5 . 6 k b and a 1.6 kb p o l y ( A ) - c o n t a i n i n g RNA. T h e p r e s e n c e of the 1.6 kb R N A is c o r r e l a t e d with the p r o d u c t i o n of the 5.6 k b viral RNA. We have not f o u n d either t h e 5 . 6 kb or 1.6 kb m e s s e n g e r R N A s in cells p r o d u c i n g the B L / K a ( B ) virus, w h i c h d o e s not c o n t a i n a 5 . 6 k b viral RNA, and the 1.6 kb R N A s h o w s v e r y little, if any, h o m o l o g y with the B L / K a ( B ) viral R N A s e q u e n c e s . It is t h e r e f o r e p o s s i b l e that the 1.6 k b R N A results from splicing of the 5 . 6 kb viral R N A

The Genome of RadLV/VL3 649

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30

FRACTIONS

Figure 7. Purification of RadLV/VL3 Virus on Low-Slope Sucrose Density Gradients Virus labeled as described in Experimental Procedures was layered on top of a preformed sucrose gradient. A 9.6 ml continuous 30-42% sucrose gradient was successively overlaid with 0.5 ml each of 25%, 20%, 10% and 5% sucrose in TNE. The gradient was centrifuged as described in Experimental Procedures and fractionated. Aliquots (20 /d) were precipitated with TCA and counted. Other aliquots (5 #1)were assayed for reverse transcriptase by a modification of the procedure of Ross et al. (1971). Incubations were performed at 30°C for 1 hr using 10/~g/ml of poly(rA):oligo(dT) as template and with 25/~Ci/ml of 3H-dTTP (New England Nuclear; 20 Ci/mmole) as substrate in a volume of 225 #1. 100/d aliquots were precipitated and counted.

in a manner analogous to the splicing event in the 8 k b g e n o m e s ( R o t h e n b e r g et al., 1 9 7 8 ) . A l t e r n a t i v e l y , it c o u l d b e d e r i v e d f r o m a n e w t y p e o f s p l i c i n g e v e n t w i t h i n t h e 8 k b R N A w h i c h is p r o m o t e d by t h e p r e s ence of the 5.6 kb genome. The 1.6 kbpoly(A)-cont a i n i n g R N A w o u l d c o n t a i n e n o u g h i n f o r m a t i o n to C o d e for a 50,000 molecular weight protein which could p l a y a r o l e in t r a n s f o r m a t i o n , but w e d o not y e t k n o w w h e t h e r t h i s R N A is p r e s e n t on B L / V L 3 p o l y r i b o somes. W e h a v e s h o w n t h a t the 5 . 6 k b R N A c o d e s f o r t h e s y n t h e s i s in vitro of a p o l y p r o t e i n o f 1 0 0 , 0 0 0 m o l e c u l a r w e i g h t c o n t a i n i n g gag a n t i g e n i c d e t e r m i n a n t s ( F i g u r e 6). P o l y p r o t e i n s with s i m i l a r s e r o l o g i c a l r e a c tivities a n d r o u g h l y s i m i l a r m o l e c u l a r w e i g h t s h a v e b e e n t r a n s l a t e d in v i t r o f r o m the g e n o m e s o f a v i a n and murine acute defective leukemia viruses (Mellon et al., 1 9 7 8 ; Witte et al., 1 9 7 8 ) . S u c h p r o t e i n s a r e a l s o f o u n d in cells t r a n s f o r m e d with t h e s e v i r u s e s ( B i s t e r , H a y m a n a n d V o g t , 1 9 7 7 ; Hu, M o s c o v i c i a n d V o g t , 1 9 7 8 ; R e y n o l d s et al., 1 9 7 8 ; Witte et al., 1 9 7 8 ) . T h e f i n d i n g o f s u c h a p r o t e i n in s e v e r a l i n d e p e n d e n t o n c o g e n i c viral s y s t e m s s t r o n g l y s u g g e s t s t h a t t h e y p l a y a r o l e in t h e o n c o g e n i c p r o c e s s . All o f t h e a c u t e d e f e c t i v e l e u k e m i a v i r u s e s s t u d i e d to d a t e c o n t a i n p a r t i a l l y d e l e t e d gag a n d env g e n e r e g i o n s w h e n c o m p a r e d with t h e i r viral h e l p e r s , with n o n h e l p e r R N A s e q u e n c e s i n s e r t e d b e t w e e n t h e s e r e g i o n s ( H u , Lai a n d V o g t , 1 9 7 9 ; S h i e l d s et al., 1 9 7 9 ) . T h e s i m i l a r i t y o f o r g a n i z a t i o n a n d e x p r e s s i o n o f R N A s e q u e n c e s at t h e 5' e n d o f t h e g e n o m e s of t h e a c u t e d e f e c t i v e

leukemia viruses and of the 5.6 kbgenome of RadLV/ VLa is s t r i k i n g . H o w e v e r , t h e n e o p l a s t i c d i s e a s e ind u c e d b y R a d L V is n o t a c u t e in o n s e t ( t h e mice develop lymphomas 3 months or more after virus inoculation), and the presence of a 100,000 molecular w e i g h t p r o t e i n with s e r o l o g i c a l p r o p e r t i e s s i m i l a r to t h a t t r a n s l a t e d f r o m the 5 . 6 k b R N A h a s y e t t o b e d e m o n s t r a t e d in t h e t r a n s f o r m e d cells. T h i s w o r k is n o w in p r o g r e s s in o u r l a b o r a t o r y . If f u r t h e r s t u d i e s establish that both the 8 and 5.6 kb genomic RNAs a r e f o u n d in all R a d L V p r e p a r a t i o n s i s o l a t e d f r o m other virus- and radiation-induced thymic lymphomas ( L i e b e r m a n et al., 1 9 7 9 ) , t h i s w o u l d s t r o n g l y s u p p o r t t h e v i e w t h a t t h e s m a l l e r g e n o m e p l a y s an e s s e n t i a l r o l e in t h e o n c o g e n i c p r o c e s s . Experimental Procedures Cells and Viruses

The BL/VL~ cell line was derived from a RadLV-induced thymic lymphoma of C57BL/Ka mouse and produces RadLV/VL3 virus (Lieberman et al., 1979). BL/Ka(B) virus was originally produced by C57/Ka embryo fibroblast cultures infected with RadLV (Decleve et al., 1976); it is maintained in permanently infected cultures of SC-1 cells (Hartley and Rowe, 1975). All cell lines were propagated in Falcon flasks in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS). To obtain large quantities of cells, RadLV/VL3 cells were grown in 3 I Bellco spinner flasks and SC-1 fibroblasts (BL/Ka(B) producers) in Coming 490 cm2 roller flasks in MEM-10% FCS supplemented with antibiotics. Virus Labeling and Purification

To minimize viral RNA degradation due to prolonged incubation of the virus at 37°C in culture medium (Cheung et al., 1972), cell culture medium was changed immediately prior to labeling and replaced with fresh MEM containing less FCS (2%). 6 I of exponentially growing BL/VL3 cells (1.5 x 106 cells per ml) were pelleted at low speed at room temperature and resuspended in 800 ml of bicarbonate-free MEM buffered with 25 mM HEPES (pH 7.4), 2% FCS, containing 25 /~Ci/ml of 3H-uridine (30 Ci/mmole, New England Nuclear). Fibreblasts producing BL/Ka(B) virus were labeled before reaching confluence using 14 rollers (1.8 x 107 cells per roller). Labeling with 3Huridine (37.5 #Ci/ml) was performed in 30 ml of MEM, 2% FCS and 25 mM HEPES per roller. After 4.5 hr of labeling, the media were collected and the viruses were isolated at 0°C. Cells and cell debris were removed by centrifugation at 400 x g for 10 min and the supernatanf was centrifuged at 10,000 x g for 20 min. Viruses were collected from the supernatant by centrifugation at 40.000 x g for 3 hr. The radioactive medium was discarded and the viral pellets were overlaid with 0.4 ml of 50 mM Tris (pH 7.4), 100 mM NaCI, 1 mM EDTA (TNE) and left on ice overnight. The viral suspensions were then gently homogenized by pipetting in 1 ml disposable plastic pipettes and the viruses were purified by sucrose equilibrium density gradients at 150,000 x g overnight at 4°C. The BL/Ka(B) virus was recovered at a density of 1.16 g/cm 3 in a 20-45% sucrose gradient in TNE. A low slope sucrose gradient was used to purify the RadLV/ VL3 virus (Figure 7). By removing particles containing degraded RNA, this gradient allowed the recovery of virus containing intact RNA, which is essential for the separation of the two RNA species present in RadLV/VLa. When virus in peak B (1.16-1.17 g/cma; Figure 7) was diluted 3 fold in TNE and centrifuged at 200,000 x g for 1 hr, 50-70% of the labeled RNA was lost into the supernatant, while lees than 10% of the RNA in peak A (1.18-1.19 g/cm3; Figure 7) or in BL/Ka(B) virus was not sedimented under the same conditions. Moreover, the RNA from peak B which was rapidly sedimented contained mainly small RNA fragments (-100 bases) when analyzed on denaturing gels (data not shown). Finally, when the proteins of

Cell 650

peak B were analyzed after labeling wit h 14C-leucine they were found to be primarily RadLV/VL3 viral proteins (data not shown). Peak B also contains very small amounts of active reverse transcriptase (Figure 7). We concluded that peak B contains primarily degraded RadLV virus, and it was discarded.

Purification and Analysis of RNA All glassware and solutions were sterilized by autoclaving before use. Virus, pelleted in TNE as described above, was resuspended in 10 mM sodium acetate (pH 5.2), 10 mM EDTA, 2% SDS and 25 Fg/ml of yeast RNA (extensively deproteinized with phenol), and the suspension was immediately homogenized by vortexing with an equal volume of phenol (saturated in 10 mM sodium acetate, 10 mM EDTA). RNA extraction was performed as described by Manteuil and Girard (1974), except that all steps were carried out at room temperature. The final aqueous phase was adjusted to 0.3 M sodium acetate (pH 6.5) and the RNA was precipitated with 2.5 vol of ethanol at -20°C. Cytoplasmic BL/VL3 cellular RNA was extracted following the technique of Palmiter (1974). Poly(A)-containing cytoplasmic RNAs were selected twice on oligo(dT)-cellulose (T-3, Collaborative Research). RNA pellets were resuspended in solutions of 8 M urea (Ultra Pure, Schwarz-Mann), 10 mM Tris-HCI (pH 7.4), 1 mM EDTA containing the indicated concentrations of NaCI. These solutions were prepared just before use and were filtered through 0.45 /~m Millipore filters. The RNAs were then incubated at 40°C for 5 min and diluted 4 fold with TNE prior to loading on a 15-30% glycerol gradient in TNE, 0.2% SDS. The RNAs were sedimented at 39K rpm at 22°C in an SW40 rotor for the indicated times. For molecular weight determinations the RNAs were electrophoresed as described by Bailey and Davidson (1976). After staining with ethidium bromide, the RNAs were visualized on a Mineralight transilluminator (254 nm Ultra-Violet Products Inc,). Photographs were taken on Polaroid 665 film through an orange filter. Gels were prepared for fluorography according to the method of Bonnet and Laskey (1974), except that acetone containing 11% PPO was used. Fluorography was carried out using Kodak XR-5 film with an intensifying screen (Dupont-Cronex) at - 70°C. RNAs to be transferred to DBM paper were first separated on agarose gels. The RNA was first denatured with glyoxal according to the method of McMaster and Carmichael (1977). After electrophoresis, the gel was stained and the glyoxal was removed by incubation in 500 ml of 50 mM NaOH, 0.5/Lg/ml ethidium bromide for 50 rain at room temperature. The gel was neutralized with two changes of 0.1 M sodium phosphate (pH 6.7) for 20 min each. The ribosomal RNA markers were readily detected under ultraviolet light and photographs were taken as described above. The gel was then rinsed twice in 20 mM sodium phosphate (pH 6.7) for 20 rain. The DBM paper was prepared according to the method of Alwine, Kemp and Stark (1977), except for the following modifications: four washes of cold water (10 min) were used after the NaNO2 treatment of the paper, followed by two rinses (10 min each) with 20 mM sodium phosphate (pH 6.7) instead of sodium borate buffer. The transfer was also carried out in 20 mM sodium phosphate. Preparation and Analysis of Strong-Stop cDNAs The procedure used was essentially the one described by Haseltine et al. (1976). After ethanol precipitation, viral RNA pellets containing purified 70S or 54S viral RNAs were rinsed with 90% ethanol and repelleted, dried in vacuo and resuspended in 25/d of 50 mM TrisHCI (pH 8.3), 10 mM MgCI2, 20 mM ,8-mercaptoethanol, 1 mM each of dGTP, dATP and dTTP and 6 #M of ~-32p-dCTP (350 Ci/mmole, Amersham). 1 #1 aliquots were precipitated with TCA and counted to determine RNA concentrations, and 6 units of purified AMV reverse transcriptase (a gift from K. Fry) were added. Reactions were performed in 0.5 ml microfuge tubes (Brinkman) and incubated for 60 rain at 46°C. The RNA templates were then hydrolyzed with 0.1 N KOH at 80°C for 15 min. The solutions were neutralized and the cDNAs were ethanol-precipitated together with 10/~g of carrier yeast RNA. The pellets were rinsed with 90% ethanol, dried in vacuo and

resuspended in 20 /~1 of 50 mM NaOH, 0.5 mM EDTA, 5 M urea, 0.025% bromphenol blue, 0.025% cyanol blue. The solutions were heated to 90°C for 15 sec and loaded on a 42 x 30 cm polyacrylamide gel (15% acrylamide, 0.5% bisacrylamide) in 7 M urea, 50 mM Tris-borate (pH 8.3), 1 mM EDTA. The electrophoresis buffer was 50 mM Tris-borate (pH 8.3), 1 mM EDTA. The gel was electrophoresed for 4-6 hr prior to loading the RNA. Electrophoresis was performed at 600 V for 17-20 hr. After electrophoresis the gel was tightly covered with Saran Wrap and autoradiographed with Kodak XR-5 Xray film.

Preparation of Randomly Primed cDNA Probes A modification of the method of Taylor, IIImensee and and Summers (1976) was used. Viral RNA pellets containing purified dimers were rinsed with 90% ethanol and dried in vacuo, suspended in 25/d of 0.1 mM EDTA and heated at 100°C for 1 min. The solutions were quickly cooled and a 1 /d aliquot was TCA-precipitated and counted. 25/d of a solution containing calf thymus DNA oligomers (500/Lg/ ml), 200 mM Tris-HCI (pH 8.1), 2 mM MgCI2, 0.28 M ,8-mercaptoethanol, 50 p.g/ml of actinomycin D (PL Biochemicals), 1 mM each of dATP, dTTP and dGTP and 12 ~M of ~-32P-dCTP (350 Ci/mmole, Amersham) was added to the RNA. Reverse transcriptase (12 U) was added and the synthesis allowed to take place for 3 hr at 37°C. The template was hydrolyzed with 0.1 N NaOH at 37°C for 1 hr. After neutralization, the cDNA was precipitated with ethanol in the presence of 20 Fg of yeast RNA carrier. The cDNA was further purified on a Sephadex G75 column equilibrated with 10 mM Tris-HCI (pH 7.4), 1 mM EDTA in a siliconized pasteur pipette. Material recovered at the void volume was reprecipitated with ethanol. The yield of DNA was approximately 10% the mass of the RNA template. Hybridization of cDNA Probes to RNA Immobilized on DBM Paper Hybridizations were performed according to the procedures of Alwine et al. (1977). 32P-labeled cDNA probes (1 x 105 to 5 × 105 cpm per ml per 30 cm 2 of paper) were hybridized to the RNA immobilized on the DBM paper for 2-3 days. In Vitro Translation of Purified RadLV/VL3 RNAs Purified RadLV/VL3 70S and 54S dimers were denatured by incubation in 8 M urea, 10 mM Tris-HCI (pH 7.4), 1 mM EDTA at 40°C for 5 rain. The solutions were then adjusted to 0.5 M NaCI and the RNAs were precipitated with 2.5 vol of ethanol. After centrifugation, the pellets were rinsed with 90% ethanol and dried in vacuo. They were resuspended in 2/d of H20 and immediately transferred to a 25 /d translation reaction using a rabbit reticulocyte lysate and 35Smethionine (New England Nuclear Translation Kit). The translations were stopped after 45 min at 37°C, Aliquots (5/d) were immediately quick-frozen and stored in liquid nitrogen. Antibody precipitations were performed in 100/d of 25 mM TrisHCI (pH 7.4), 0.2 M NaCI, 1 mM EDTA and 0.1% Triton X-100 (TENT). Samples were precipitated with goat antisera against Rauscher or Gross viral proteins (a gift from M. Strand and J. T. August) for 30 rain at room temperature followed by 1 hr at 4°C. When indicated, 2 /tg of RadLV/VL3 P30 protein or 1 /~g of RadLV/VL3 Pt 2 protein (gifts from J, N. Ihle) were added and the incubation continued overnight. After centrifugation at 12,000 × g for 30 rain at 4°C, the pellets were rinsed with 50/d of cold TENT with vortexing, repelleted and resuspended in 30/~1 of 10% glycerol, 0.7 M/~-mercaptoethanol, 3% SDS, 62 mM Tris-HCI (pH 6.8). Samples were heated to 90°C for 3 rain. Bromophenol blue tracking dye was added to 0.05% and the samples were loaded on top of 5-15% acrylamide-SDS gels prepared according to the method of Laemmli (1970) in Tris-glycine buffer. A 5% acrylamide stacking gel was used. Gels were 9 or 16 cm in length. Samples were electrephoresed for 2 hr on short gels or for 6 hr on long gels at 150 V. Gels were then stained in 0,05% Coomassie blue in methanol:acetic aeid:H20 (5:1:5) for 2 hr at 37°C, destained in 7.5% acetic acid for 2 hr at 37°C with four changes of solution and prepared for fluorography according to the method of Bonner and Laskey (1974). Fluorography was carried out as described above. Stained protein markers were still visible on the dried gel and their

The Genome of RadLV/VL3 651

positions were compared with the bands on the fluorogram for molecular weight determination of the translated products.

Decleve, A., Lieberman, M., Ihle, J. N. and Kaplan, H. S. (1976). Biological and serological characterization of radiation leukemia virus. Proc. Nat. Acad. Sci. USA 73, 4675-4679.

Acknowledgments

Decleve, A., Lieberman, M., Ihle, J. N., Rosenthal, P. N., Lung, M. L. and Kaplan, H. S. (1978). Physical-chemical, biological and serological properties of a leukemogenic virus isolated from cultured RadLVinduced lymphomas of C57BL/Ka mice. Virology 90, 23-35.

We would like to thank Dr. Mette Strand for her gift of antibodies and for her helpful advice; Dr. Kirk Fry for his help and gift of AMV reverse transcriptase; Dr. Tao-shih Hsieh for his gift of DNA size markers and for help with the high resolution gels; James Williams and Joan Kojola for growing cells for us; and Dr. Douglas Brutlag for help with the manuscript. These studies were supported by the Joseph Edward Luetje Memorial Fund for Lymphoma Research and, more recently, by a research grant from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received September 6, 1979; revised October 9, 1979

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scription of RNA into DNA by avian sarcoma virus polymerase. Biochim. Biophys. Acta 442, 324-330. , ~: _ Weiss, S. R., Varmus, H. E. and Bishop, J. M. (1977). The size and genetic composition of virus-specific RNAs in the cytoplasm of cells producing avian sarcoma-leukosis viruses. Cell 12, 983-992. Witte, O. N., Rosenberg, N., Paskind, M., Shields, A. and Baltimore, D. (1978). Identification of an Abelson murine leukemia virus encoded protein present in transformed fibroblast and lymphoid cells. Proc. Nat. Acad. Sci. USA 75, 2488-2492. Note Added in Proof

Recent results obtained in collaboration with Drs. Steven Goff and David Baltimore demonstrate that the 8 kb and 5.6 kb RadLV/VL3 RNAs do not contain Abelson-specific viral sequences. If the 5.6 kb RadLV RNA codes for a transforming protein, this protein must therefore differ from the Abelson-transforming protein.