Vol. 64, No. 3

JOURNAL OF VIROLOGY, Mar. 1990, p. 1378-1382

0022-538X/90/031378-05$02.00/0 Copyright C 1990, American Society for Microbiology

Reversion of Thermosensitive Splicing Defect of Moloney Murine Sarcoma Virus tsllO by Oversplicing of Viral RNA RICHARD HAMELIN,'* NICOLE HONORE,2 DINA SERGIESCU,3 BALRAJ SINGH,4 JACQUELINE GERFAUX,3 AND RALPH B. ARLINGHAUS4 Institut d'Oncologie Cellulaire et Moleculaire Humaine, 129 Boulevard de Stalingrad, 93000 Bobigny,' INSERM U-248, Faculte de Medecine Lariboisiere-Saint Louis, 75010 Paris,2 and INSERM U43, H6pital Saint-Vincent-de-Paul, 75014 Paris,3 France, and Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 770304 Received 11 July 1989/Accepted 8 November 1989

Moloney murine sarcoma virus tsllO possesses a thermosensitive splicing defect. By continuously growing nonproducer cells at the nonpermissive temperature, a new class of revertant cells, termed 6m3, that had lost the thermosensitive splicing defect was produced, and six distinct clones were selected. These cell clones were transformed at either permissive or restrictive temperatures. Unlike parental 6m2 cells, which contain two virus-specific RNA species of 4.0 and 3.5 kilobases (kb) at temperatures permissive for transformation, the 3.5-kb RNA was the only virus-specific RNA species detected in 6m3 clones. No new v-mos-containing DNA fragment was observed in Southern blot analysis of these cell clones compared with parental 6m2 cells, indicating that the 3.5-kb RNA was a splicing product rather than a direct transcript. Moreover, these cells expressed P85gag-mOs but not P58gag at any temperature. The reversion of the phenotype in 6m3 cell clones appears to be the result of a selective loss of the temperature sensitivity of the splicing reaction, without affecting the thermosensitivity of the protein kinase activity. This change also appears to alter the mechanism regulating the efficiency of the genomic RNA-splicing reaction.

splicing requirement. Two other MuSV tsllO revertant cell lines (54-5A4 and 204-3) also appear to be transformed at all growth temperatures from 28 to 39°C (12, 35). They contain only PlO'ag-mos and a 4.0-kb RNA species that is never spliced (12, 35). It has been shown that a 5-base deletion at the intron-exon border of the 3' splice site has at the same time eliminated the possibility of a splice and produced a continuous open reading frame from gag to mos, allowing the translation of a P1009'1~°-transforming protein at either growth temperature (7). A third type of revertant has been obtained by nickel chloride treatment of 6m2 cells (4). The nickel-induced revertants stably maintain the transformed phenotype at 39°C; they express 3.5-kb RNA as well as unspliced 4.0-kb genomic RNA together with P58gag and P85gag-mos (and its associated serine kinase activity) at this temperature. In these cell lines, the reversion is explained by a loss of the thermosensitivity of the splicing reaction. Similar revertants have been obtained by N-nitroso-N-methylurea treatment of 6m2 cells, whereas revertants of the second type were obtained by cadmium chloride and sodium chromate treatment (5). In this report, we describe a fourth type of MuSV tsllO revertant obtained by continuously growing 6m2 cells at the nonpermissive temperature. 6m2 cells, which contain the MuSV tsllO-splicing mutant, are normally grown at 33°C. When shifted to 37 to 39°C, their general morphology is changed and they grow very slowly compared with growth at the nonrestrictive temperature. Colonies of round cells appear at various places and within a few passages (3 to 4 weeks) completely invade the culture. Continuous growth of 6m2 cells at 39 and 37°C produced two different populations of cells called 6m3 and 6m4, respectively. Both new cell lines appeared morphologically transformed at any growth temperature and grew slightly better at 37°C than at 33°C; 6m3 and 6m4 cells grew faster than 6m2 cells at 37°C, while all three cell lines grew at approximately the same rate at 33°C (data not shown).

Murine sarcoma virus tsllO (MuSV tsllO) is a conditionally transformation-defective retrovirus obtained by UV irradiation of MuSV 349 virions, a subclone of MuSV 124 (6). A nonproductively MuSV tsllO-infected normal rat kidney cell line (the 6m2 cell line) is morphologically transformed when grown at 28 to 33°C but not at 37 to 39°C (2, 8, 23). At permissive temperatures, 6m2 cells contain two virus-specific proteins termed P58gag and P85gag-mos, but only P589'9 is detected at 39°C (16, 17). When present, p859a'mos has an associated serine-threonine protein kinase activity that is responsible for the transformation of 6m2 cells (22, 23). Two virus-specific RNA species of 4.0 and 3.5 kilobases (kb) encoding P58gag and P85gag-mos respectively, are present in 6m2 cells grown at 28 or 33°C (8, 12, 20, 27). Splicing of the 4.0-kb RNA produces the 3.5-kb RNA (8, 12). The effect of the splice event is to fuse gag and mos genes in frame, resulting in a continuous open reading frame coding for P85gag-mos. No 3.5-kb RNA can be detected at the restrictive temperature of 39°C, and it has been demonstrated that the splicing event is thermosensitive (8, 12). We have also shown that this splicing defect cannot be complemented by superinfection of 6m2 cells with a helper virus (12) and that is encoded by the virus rather than by the cells

(14).

Several cell lines containing MuSV tsllO revertants have already been described. The NRK-206-21C cell line, generated by superinfection of 6m2 cells with Moloney murine leukemia virus, produces a 3.5-kb RNA species at 39°C as well as at 28°C and contains proviral DNAs corresponding to the two viral RNA species (12, 28). In these cells, the splicing of the 4.0-kb RNA appears to remain thermosensitive. However, additional 3.5-kb RNA can be independently transcribed from its own proviral DNA at either permissive or nonpermissive temperatures, thus bypassing the usual *

Corresponding author. 1378

VOL. 64, 1990

1

NOTES

2 3

4

5 6 7 8 9 10

1 2 3

4 5 6 78 91011 12

F,*,

-*

A

W

13

a

68340 kb 3 5 kb-

1379

K28s

-495 -

.AS s

450_

qwe4

o

I L~~~~~~~~ FIG. 1. Virus-specific RNA in 6m3 clones. Northern blot analysis of total RNAs extracted from 6m2 cells grown at 28°C (lane 1), 33°C (lane 2), or 39°C (lane 3); from C-23, C-24, C-28, C-52, C-53, and C-58 6m3 clones (lanes 4, 5, 6, 7, 8, and 9, respectively) grown at 37°C; and from 6mT cells (lane 10).

When five nude mice were injected subcutaneously with 6m3 cells, all five developed tumors after 2 weeks. Only one of five nude mice injected with 6m2 cells developed a tumor after 4 months. This last tumor was established in vitro (and named 6mT) and showed a fibroblastic morphology. When these 6mT cells were injected into five nude mice, no tumors were seen after 6 months. 6m3 cells were cloned by both dilution and agar growth (25), and six different clones were selected and analyzed in detail. All displayed the same transformed phenotype as 6m3 cells at 37°C (data not shown). It has already been established that 6m2 cells contain two virus-specific RNA species of 4.0 and 3.5 kb when grown at permissive temperatures of 28 to 33°C, but only the 4.0-kb genomic RNA is detected at the restrictive temperatures of 37 to 39°C (8, 12, 20). This is shown in Fig. 1. Total RNAs extracted by the hot-phenol procedure (15) from 6m2 cells grown at various temperatures were analyzed by the Northern (RNA) blot method as already described (13, 38) and revealed by hybridization with a mos-specific probe (20) (Fig. 1, lanes 1 to 3). The only mos-containing RNA species detected in 6m3 clones (Fig. 1, lanes 4 to 9) corresponded in size to the 3.5-kb spliced RNA present in 6m2 cells grown at the permissive temperatures. Absence of the 4.0-kb RNA species in 6m3 clones was further documented by Si protection experiments with the BglII-KpnI 683-base-pair DNA fragment as previously described (14, 29). A total of 495 and 450 bases of this DNA fragment were protected against Si hydrolysis by the 4.0- and 3.5-kb RNA species, respectively, when present in 6m2 cells (Fig. 2, lanes 1 to 3). Only the 450-base DNA fragment was protected by RNA extracted from 6m3 clones (Fig. 2, lanes 6 to 11) and from uncloned 6m3 cells at passage 45 (Fig. 2, lanes 4 and 5). Finally, no mos-containing RNA was detected either by Northern blot (Fig. 1, lane 10) or by the Si protection experiment (Fig. 2, lane 12) in 6mT cells. To substantiate the RNA data presented above, we analyzed the protein products of one of the cloned cell lines (C-28) derived from the 6m3 cells. The C-28 cells were grown at 37°C and pulse labeled with [3H]leucine (40 to 60 Ci/mmol; Dupont, NEN Research Products, Boston, Mass.) at 500 p.Ci/ml in Earle balanced salt solution for 30 min. The cell extracts were immunoprecipitated with various anti-gag

FIG. 2. S1 nuclease analysis of the 3' deletion border in tsllO MuSV RNA in 6m3 clones. Cellular RNAs were hybridized with a 5'-end-labeled BglIl-KpnI 683-base-pair DNA fragment and processed as described previously (14). The cellular RNAs were extracted from 6m2 cells grown at 28°C (lane 1), 33°C (lane 2), or 39°C (lane 3); from 6m3 cells (passage 45) grown at 33°C (lane 4) or 37°C (lane 5); from C-23, C-24, C-28, C-52, C-53, and C-58 6m3 clones (lanes 6, 7, 8, 9, 10, and 11, respectively) grown at 37°C; and from 6mT cells grown at 37°C (lane 12). Lane 13 was done without added RNA. Fragment sizes (in bases) are indicated.

antibodies and the anti-mos antibody (37-55) (9) (Fig. 3, lanes 1 to 5). 6m2 cells labeled at 30°C were analyzed in parallel (Fig. 3, lanes 6 to 10). The P85gagn-os protein, which is recognized by anti-piS, anti-p12, anti-p30, and anti-mos antibodies, was detected in both 6m2 cells and C-28 cells. However, the P58gag protein, which is encoded by the 4.0-kb viral RNA, was easily detected in 6m2 cells but was undetectable in C-28 cells. We failed to see any trace of P58gag in the C-28 cells, even at a much longer exposure. We next determined whether P58gag could be found in the C-28 cells via phosphorylation by P85za9r-ns in the immune complex reaction. Both P85gag-ms and P58gag could be detected by this method in 6m2 cells maintained at 30°C for 24 h (Fig. 4A). While P85gag-rns autophosphorylation was readily visible, no phosphorylated P58gag was observed in the immune complexes from the C-28 cells grown at 37°C with either of the anti-gag antibodies (Fig. 4B). To determine whether the protein kinase activity associated with P85gag-mos was thermosensitive, we performed the in vitro kinase assay after shifting the C-28 cells to 30 or 39°C for 24 h. A large decrease in the autophosphorylation of P85gagmos was observed at 39°C (compare lanes 1, 6, and 7 in Fig. 4B). As expected, 6m2 cells that had been shifted to 39°C for 24 h lacked detectable P85gag-mos as measured by the in vitro kinase reaction (Fig. 4A, lane 7). In the revertant C-28 clone, the effect of the 39°C shift seen in the in vitro kinase assay was not as dramatic as in 6m2 cells. This can be explained by the continuous production of P85gag-mos in C-28 cells maintained at 39°C and the lack of P85gag-mos synthesis in 6m2 cells under these conditions. However, 6m3 cells grown at restrictive temperatures maintained the transformed phenotype, undoubtedly because of an overproduction of a heatlabile, but still functional, transforming protein. A similar conclusion was reached with nickel-treated 6m2 cells, which also continue to express thermosensitive P85gag-mos at re-

1380

J. VIROL.

NOTES

1

2

4

3

5

6

7

8

9

10

95 -

-

p85gag-nos

P58gag

4336_

FIG. 3. Detection of viral proteins in C-28 and 6m2 cells. The C-28 cells were grown and labeled at 37°C for 30 min with [3H]leucine (0.5 mCi/ml). The subconfluent 6m2 cells, which were grown at 33°C, were shifted to 30°C for 24 h before labeling. The cell extracts were prepared as described previously (30), and immunoprecipitation was done with anti-gag or anti-mos antibodies. Lanes: 1 to 5, C-28 cells; 6 to 10, 6m2 cells. Antibodies: lanes 1 and 6, anti-mos (37-55); lanes 2 and 7, peptide-blocked anti-mos (37-55) antibodies; lanes 3 and 8, anti-p15; lanes 4 and 9, anti-p12; lanes S and 10, anti-p30. The immunoprecipitated proteins were resolved in an 8% sodium dodecyl sulfate-polyacrylamide gel (1) and visualized by fluorography (19). On the left are shown the molecular size standards (in kilodaltons). The P58gag band is identified by an asterisk.

strictive temperatures yet maintain the transformed phenotype (4). It has been shown previously that 6m2 cells contain only one tsllO provirus which is translated into two viral RNA species, one full length and one spliced subgenomic (12, 28). In reverted 206-2IC cells, we have detected a proviral DNA corresponding in size to the subgenomic mRNA species (12, 28), and it was thus of interest to analyze proviral DNAs in 6m3 and 6m4 cells to eliminate the possible translation of a 3.5-kb subgenomic RNA produced directly from a shorter provirus. DNAs extracted from 6m3 clones and from 6m4

A

1

cells were analyzed by the Southern blot method (Fig. 5). There was no difference in patterns observed among any of these cell lines and the 6m2 cells with the restriction enzymes used in this experiment. A more complete comparison between 6m2 cells and the C28 clone was made by using nine different restriction enzymes, and again no differences were found (data not shown). In the same Southern blot, we analyzed DNA extracted from the 6mT cell line. The mos-containing DNA fragments, when 6mT DNA was digested with BamHI, BamHI plus Sad, and Hindlll, are shown in Fig. 5, lanes A8, B8, and

2 34 5 67

B.

1

2 3 4 5 6

7

nls-mqql 1s__ 95

p85gag-mos

P58gag

P,Om

30

3339.

-M

.

37

39

-

p85gag-mos

.(

55 43

.e

36 30

FIG. 4. In vitro kinase assay with the viral proteins of 6m2 and C-28 cells. Immune complex kinase assays were performed on 6m2 cells (A) or C-28 cells (B) as described previously (24). (A) 6m2 cells were grown at 33°C (lane 6) and shifted to either 30°C (lanes 1 to 5) or 39°C (lane 7) for 24 h. Antibodies used for immunoprecipitation were anti-mos (37-55) (lane 1); peptide-blocked anti-mos (37-55) antibodies (lane 2); anti-plS (lane 3); anti-p12 (lane 4); anti-p30 (lanes 5, 6, and 7). (B) C-28 cells were grown at 37°C (lanes 2 to 6) and shifted to either 30°C (lane 1) or 39°C (lane 7). Antibodies used were anti-mos (37-55) (lane 2); the peptide-blocked anti-mos (37-55) antibodies (lane 3); anti-plS (lane 4); anti-p12 (lane 5); anti-p30 (lanes 1, 6, and 7). The in vitro-phosphorylated proteins were resolved in a sodium dodecyl sulfate-8% polyacrylamide gel and visualized by autoradiography with preflashed X-omat RP film (Kodak). Between the panels are shown the molecular size standards (in kilodaltons).

NOTES

VOL. 64, 1990

A

wlhn4M 1

2 3

5 6

4

7

B

8

qX

10,

lti

.....'

...

1

4_m 1

2

*

6

3 J4

10

-w

c

wV

VV

I0 0

b,VV

towe

FIG. 5. Mos-containing DNA in 6m3 clones, 6m4, and 6mT cells. High-molecular-weight DNA was digested with BamHI (A), BamHI-SacI (B), and Hindlll (C), analyzed on a 0.7% agarose gel, transferred to nitrocellulose (33), and hybridized to a 32P-labeled mos probe (20). The DNAs were extracted from C-58 (lanes 1), C-53 (lanes 2), C-52 (lanes 3), C-28 (lanes 4), C-24 (lanes 5), C-23 (lanes 6), 204-2F6 (lanes 7), 6mT (lanes 8), 6m4 (lanes 9), 6m3 (lanes 10), and 6m2 (lanes 11) cells. Sizes of the HindlIl-cleaved A bacteriophage fragments (in kilobases) are indicated at the sides of the panels.

C8, respectively. In each case, only one fragment was revealed by hybridization with the mos-specific probe. We propose that this fragment is the mouse c-mos, since it differs in size from the rat c-mos detected in 6m2 and other MuSV tsllO-infected NRK cells. The temperature-sensitive phenotype of transformation caused by MuSV tsllO is the result of two conditional defects encoded by the mutant virus. One defect concerns the heat lability of the transforming protein, P85gag-mos (34) and the related thermosensitivity of its associated serinethreonine protein kinase (23). The second conditional defect is the thermosensitivity of the splicing of the subgenomic mRNA encoding the transforming protein P85gag-ms (8, 12,

1381

20). Three mechanisms of reversion of MuSV tsllO have been described in previous reports: integration of a subgenomic provirus that can be directly transcribed in a moscoding mRNA without the need of a splicing reaction (type I) (12, 28); deletion in the splice acceptor site inhibiting splicing and fusing in frame the gag and mos genes (type II) (7, 12, 35); and loss of the thermosensitivity of the splicing reaction (type III) (4, 5). We describe in this report a new type of revertant of MuSV tsllO that is clearly different from the three other types. It is not a type I revertant because there is only one full-length provirus integrated in the cellular genome. It synthesizes P85gaz-mos instead of plOOaag"-rs and is thus different from type II revertants. Finally, the 3.5-kb species is the only viral RNA species detected at any growth temperature, distinguishing it from type III revertants. The mechanism of reversion of the MuSV tsllO revertants described in this report is a temperature-independant oversplicing of the viral RNA leading to a complete disappearance of genomic RNA and its translated gag product in reverted cells. It must be ruled out, however, that accelerated degradation of 4.0-kb RNA remaining after the splicing reaction is also taking place in this fourth type of revertant. Eucaryotic transcripts either do not contain intervening sequences and do not require splicing or, more often, contain introns that are removed completely so that no unspliced pre-mRNA is found in the cytoplasm (for reviews, see references 10, 11, 31, and 32). In some cases, genes that are expressed through alternative splicing must have an additional regulatory element(s) acting in trans or in cis or both. Retroviral pre-mRNA splicing appears to be a particular form of alternative splicing; unspliced RNA is used both as genomic RNA and as mRNA for gag and pol genes, whereas spliced RNAs are used as mRNAs for the 3'-located viral genes. Consensus sequences for splicing are conserved in retroviral RNAs and, although a possible inefficient recognition of these sequences by cellular splicing factors is not completely ruled out, it is probable that cis- or trans-acting factors have an important role in the maintenance of the proper balance between spliced and unspliced retroviral RNA. cis-acting sequences have been defined in avian retroviruses (3, 21, 26, 36, 37). These appear to be mostly negative regulatory elements, because when they are removed by deletion or modified by insertion, more mRNA is

spliced. In murine retroviruses, negative cis-acting regulatory ele-

regions of Moloney murine leukemia virus were found to be required for efficient splicing (18). One of these regions includes the branch-point signal and the other one is within a gag-coding ments have not been shown. In contrast, two

sequence. We propose that MuSV tsllO RNA possesses a strong negative cis-acting regulatory element that is partly overridden in cells maintained at permissive temperatures. In the MuSV tsllO revertants present in 6m3 and 6m4 cells, the negative regulatory cis-acting element is mutated and no longer active, so that the viral RNA is overspliced. Further molecular studies on the revertant described here should be useful in understanding the regulation of retrovirus splicing. We thank J. Wybier-Franqui and D. Brouty-Boye for performing the animal experiments and D. Siegel for critical reading of the

manuscript.

This work was supported in part by grant 6616 from the Association pour la Recherche sur le Cancer (R.H.), by Public Health Service grants CA 45217 and CA 16672 from the National Institutes of Health (R.B.A.), and by grant G1118 from the Robert A. Welch Foundation (R.B.A.).

1382

1.

2. 3. 4.

5.

6.

7.

8.

9.

10. 11.

12.

13.

14. 15.

16.

17.

18.

19.

J. VIROL.

NOTES

LITERATURE CITED Arcement, L. J., W. L. Karshin, R. B. Naso, G. A. Jamjoom, and R. B. Arlinghaus. 1976. Biosynthesis of Rauscher leukemia virus proteins: presence of p30 and envelope p15 sequences in precursor polyproteins. Virology 69:763-774. Arlinghaus, R. B. 1985. The tsllO Moloney mouse sarcoma virus system: gag-mos gene products and cellular transformation. J. Gen. Virol. 66:1845-1853. Arrigo, S., and K. Beemon. 1988. Regulation of Rous sarcoma virus RNA splicing and stability. Mol. Cell. Biol. 8:4858-4867. Biggart, N. W., G. E. Gallick, and E. C. Murphy, Jr. 1987. Nickel-induced heritable alterations in retroviral transforming gene expression. J. Virol. 61:2378-2388. Biggart, N. W., and E. C. Murphy, Jr. 1988. Analysis of metal-induced mutations altering the expression or structure of a retroviral gene in a mammalian cell line. Mutat. Res. 198: 115-129. Blair, D. G., M. A. Hull, and E. A. Finch. 1979. The isolation and preliminary characterization of temperature-sensitive transformation mutants of Moloney sarcoma virus. Virology 95: 303-316. Cizdziel, P. E., M. A. Nash, D. G. Blair, and E. C. Murphy, Jr. 1986. Molecular basis underlying phenotypic revertants of Moloney murine sarcoma virus MuSV tsllO. J. Virol. 57: 310-317. Gallick, G. E., R. Hamelin, S. Maxwell, D. Duyka, and R. B. Arlinghaus. 1984. The gag-mos hybrid protein of tsllO Moloney murine sarcoma virus: variation of gene expression with temperature. Virology 139:366-374. Gallick, G. E., J. T. Sparrow, B. Singh, S. A. Maxwell, L. H. Stanker, and R. B. Arlinghaus. 1985. Recognition of mos-related proteins with an antiserum to a peptide of the v-mos gene product. J. Gen. Virol. 66:945-955. Green, M. R. 1986. Pre-mRNA splicing. Annu. Rev. Genet. 20:671-708. Hamelin, R. 1988. Retroviral genome splicing, p. 95-140. In M. Crepin (ed.), Research in retroviruses and oncogenes. INSERM/John Libbey Eurotext, Ltd., Montrouge, France. Hamelin, R., B. L. Brizzard, M. A. Nash, E. C. Murphy, Jr., and R. B. Arlinghaus. 1985. Temperature-sensitive viral RNA expression in Moloney murine sarcoma virus tsllO-infected cells. J. Virol. 53:616-623. Hamelin, R., J. Devaux, N. Honore, M. A. Auger-Buendia, and A. Tavitian. 1983. Characterization of viral RNA in cells transformed by various isolates of Moloney murine sarcoma virus. J. Gen. Virol. 64:2057-2062. Hamelin, R., K. Kabat, D. Blair, and R. B. Arlinghaus. 1986. Temperature-sensitive splicing defect of tsllO Moloney murine sarcoma virus is virus encoded. J. Virol. 57:301-309. Hamelin, R., C. J. Larsen, and A. Tavitian. 1973. Effects of actinomycin D, toyocamycin and cycloheximide on the synthesis of low molecular weight nuclear RNA in HeLa cells. Eur. J. Biochem. 35:350-356. Horn, J. P., T. G. Wood, D. G. Blair, and R. B. Arlinghaus. 1980. Partial characterization of a Moloney murine sarcoma virus 85,000 dalton polypeptide whose expression correlates with the transformed phenotype in cells infected with a temperature-sensitive mutant virus. Virology 105:516-525. Horn, J. P., T. G. Wood, E. C. Murphy, D. G. Blair, and R. B. Arlinghaus. 1981. A selective temperature-sensitive defect in viral RNA. Expression in cells infected with a ts transformation mutant of Murine sarcoma virus. Cell 25:37-46. Hwang, L. H. S., J. Park, and E. Gilboa. 1984. Role of intron-contained sequences in formation of Moloney murine leukemia virus env mRNA. Mol. Cell. Biol. 4:2289-2297. Jamjoom, G. A., R. B. Naso, and R. B. Arlinghaus. 1977.

20.

21.

22.

23. 24.

25. 26.

27.

28.

29.

30.

31. 32.

33. 34.

35.

36. 37.

38.

Further characterization of intracellular precursor proteins of Rauscher leukemia virus. Virology 78:11-34. Junghans, R. P., E. C. Murphy, Jr., and R. B. Arlinghaus. 1982. Electron microscopic analysis of tsllO Moloney murine sarcoma virus, a variant of wild-type virus with two RNAs containing large deletions. J. Mol. Biol. 161:229-255. Katz, R. A., M. Kotler, and A. M. Skalka. 1988. cis-acting intron mutations affect the efficiency of avian retroviral splicing: implication for mechanisms of control. J. Virol. 62:2686-2695. Kloetzer, W. S., S. A. Maxwell, and R. B. Arlinghaus. 1983. P85gag-mos encoded by tsllO Moloney murine sarcoma virus has an associated protein kinase activity. Proc. Natl. Acad. Sci. USA 80:412-416. Kloetzer, W. S., S. A. Maxwell, and R. B. Arlinghaus. 1984. Further characterization of the P85gas-mos associated protein kinase activity. Virology 138:143-155. Maxwell, S. A., and R. B. Arlinghaus. 1985. Serine kinase activity associated with Moloney murine sarcoma virus-124encoded P37mos. Virology 143:321-333. McPherson, I., and L. Montagnier. 1964. Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23:291-294. Miller, C. K., J. E. Embretson, and H. M. Temin. 1988. Transforming viruses spontaneously arise from nontransforming reticuloendotheliosis virus strain T-derived viruses as a result of increased accumulation of spliced viral RNA. J. Virol. 62:1219-1226. Murphy, E. C., Jr., and R. B. Arlinghaus. 1982. Comparative tryptic peptide analysis of candidate P85gag-mos of tsllO Moloney murine sarcoma virus and P38-P23 mos gene related proteins of wild type virus. Virology 121:372-383. Nash, M., B. L. Brizzard, J. L. Wong, and E. C. Murphy, Jr. 1985. Murine sarcoma virus tsllO RNA transcripts: origin from a single proviral DNA and sequence of the gag-mos junctions in both the precursor and spliced viral RNAs. J. Virol. 53:624-633. Nash, M., N. V. Brown, J. L. Wong, R. B. Arlinghaus, and E. C. Murphy, Jr. 1984. S1 nuclease mapping of viral RNAs from a temperature-sensitive transformation mutant of murine sarcoma virus. J. Virol. 50:478-488. Naso, R. B., L. J. Arcement, and R. B. Arlinghaus. 1975. Biosynthesis of Rauscher leukemia viral proteins. Cell 4:31-36. Padgett, R. A., P. J. Grabowski, M. M. Konarska, S. Seiler, and P. A. Sharp. 1986. Splicing of messenger RNA precursors. Annu. Rev. Biochem. 55:1119-1150. Sharp, P. A. 1987. Splicing of messenger RNA precursors. Science 235:766-771. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Stanker, L. H., G. E. Gallick, J. P. Horn, and R. B. Arlinghaus. 1983. P855a5-mOS encoded by tsllO Moloney murine sarcoma virus: rapid turnover at the restrictive temperature. J. Gen. Virol. 64:2203-2211. Stanker, L. H., J. P. Horn, G. E. Gallick, W. S. Kloetzer, E. C. Murphy, Jr., D. G. Blair, and R. B. Arlinghaus. gag-mos polyproteins encoded by variants of the Moloney strain of mouse sarcoma virus. Virology 126:336-347. Stoltzfus, C. M., and S. J. Fogarty. 1989. Multiple regions in the Rous sarcoma virus src gene intron act in cis to affect the accumulation of unspliced RNA. J. Virol. 63:1669-1676. Stoltzfus, C. M., S. K. Lorenzen, and S. L. Berberich. 1987. Noncoding region between the env and src genes of Rous sarcoma virus influences splicing efficiency at the src gene 3' splice site. J. Virol. 61:177-184. Thomas, P. S. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77:5201-5205.