Loss of Proviral DNA Sequences in a Revertant of Kirsten Sarcoma Virus-transformed Murine Fibroblasts

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Leo Hensley
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1 J. gen. Virol. (I979), 44, Printed in Great Britain Loss of Proviral DNA Sequences in a Revertant of Kirsten Sarcoma Virus-transformed Murine Fibroblasts (Accepted 22 February I979) SUMMARY A previously described revertant cell line (K-BALB SR1212), derived as a single cell clone from a clonal line of murine fibroblasts (K-BALB) transformed by a nonproductive infection with the Kirsten strain of murine sarcoma virus, has normal morphology and growth kinetics and, unlike the transformed parent cell line, lacks a sarcoma virus that can be rescued. We report here that this reversion correlates with low to undetectable levels of expression of cellular Ki-MSV-specific RNA and a reduction of proviral sequences in the cell DNA to a level equivalent to that found in the uninfected BALB cells with a normal phenotype. The data indicate that phenotypic reversion has occurred as a consequence of the loss of part or all of the sarcoma provirus, either by chromosomal rearrangement or provirus excision. Cultured murine fibroblasts (K-BALB; Aaronson & Weaver, I970 transformed by nonproductive infection of BALB-C/3T 3 cells with a replication-defective sarcoma virus component of Kirsten murine sarcoma-helper virus complex (Ki-MSV-MuLV) produce no detectable extracellular virus and little or no virus protein (Wu et al. 1976; Reitz et al. 1977), yet express high levels of virus-specifc RNA (Benveniste & Scolnick, I973; Wu et al I974) and contain a complete proviral DNA copy for the sarcoma virus, which can be rescued by superinfection with a helper virus (Aaronson & Weaver, I97I ). These transformed cells revert in culture, with a frequency of Io -3 to lo -5, to a normal phenotype with respect to morphology, malignancy in mice, lectin agglutinibility and sugar transport (Stephenson et al. I973; Nomura et al. I974). Some subclones of these revertant cells contain a rescuable sarcoma virus and some do not. Our results, described below, indicate that one of these revertant subclones (described by Nomura et al. 1974), which lacks a rescuable sarcoma virus, expresses little or no virus-specific RNA and has lost at least some of the provirus. Cells used in this study include: (I) a clonal line derived from normal BALB-C/3T3 murine fibroblasts (A3I; Aaronson & Weaver, I97I); (2) a clonal derivative of the A3I line, K-BALB (or KA3I; Aaronson & Weaver, I97I), transformed upon nonproductive infection with Ki-MSV; (3) a subclone or K-BALB which lacks rescuable sarcoma virus (K-BALB SR1212; Nomura et al. 1974); and (4) normal rat kidney cells (NRK; Duc-Nguyen et al. I966) infected with and producing Ki-MSV-MuLV. The virus used as a source of RNA and complementary DNA (cdna) in this study is the TR-NRK strain of Ki-MSV-MuLV which contains a IO- to Ioo-fold excess of MSV over MuLV (Maisel et al 1973). Cells were grown and harvested as previously described (Wu et al. I974). Induction experiments with 5'-iodo-2'deoxyuridine (IdUrd) were performed as described elsewhere (Wu et al. 1974). Virus was from Electronucleonics, Inc., Bethesda, Md. U.S.A. Previously described methods were used to prepare nuclear DNA (Reitz et al. I976), cytoplasmic RNA (Wu et al. I974), virus 7oS RNA (Wu et al. I974), and virus SH-cDNA (Wu et al. 1974). Virus RNA was labelled with lesi to a specific activity of Io 8 ct/min//zg. Virus 3H-cDNA was prepared at a specific activity of 2 lo T ct/min//zg. Virus cdna was also prepared in identical reactions but with concentrations of unlabelled deoxyribonucleo- oo /79/oooo-35o8 $02.00 (~) 1979 SGM

2 246 Short communications _ < Z? 6O 100 I 1 I i0 (10-2) 101(10-1) 10z(10 ) Crt (tool.s/l) Fig. ~. Hybridization of Ki-MSV ~H-cDNA to cell RNA. Ki-MSV 3H-cDNA was hybridized to the indicated RNA as described in Methods. Crt values in parentheses refer to virus 7oS RNA. -, A31 cytoplasmic RNA; A-A, K-BALB revertant SR. 121~ cytoplasmic RNA; - (3, K-BALB cytoplasmic RNA; ~k-a, Ki-MSV 7oS RNA. side triphosphates equal to that of the 3H-labelled precursors and used to test the extent of transcription. Virus cdna prepared in this manner protected 95 ~o of 3H-uridine-labelled virus RNA (2 t@ ct/min//zg) at a cdna-rna weight ratio of 4: I. Hybridization of virus 3H-cDNA to cell RNA was carried out to indicated C~t values (Birnsteil et al ) in hybridization reactions in 5O)/o formamide- 3 SSC (I SSC = o'i5 M-NaC1, o'oi5 M-sodium citrate, ph 7) at 37 C using 5oo ct/min 3H-cDNA per time point and a total of Ioo to 2oo/zg cell RNA or I #g virus RNA in a vol. of 8o to I2O #1. Hybridization of 125I-virus RNA to cell DNA was carried out to the indicated Cot using at least a lot-fold weight excess of DNA over RNA and annealing at 65 C in o'4 M-phosphate buffer, ph 6.8. Hybridization of 3H-cDNA was assayed by digestion with SI nuclease and 125I-RNA by digestion with pancreatic ribonuclease, both as described previously (Reitz et al. I976 ). As previously reported by Nomura et al (I974), there is little or no detectable release of reverse transcriptase into the media of K-BALB SR 1~12 cells treated with IdUrd. When IdUrd-treated revertant cells were also treated with dexamethasone, which enhances virus production from K-BALB cells (Wu et al. I974), a very low and somewhat late peak of media reverse transeriptase activity was observed with the revertant cells. This late peak in the case of K-BALB cells represents the N-tropic endogenous BALB virus production (Stephenson & Aaronson, i972; Aaronson & Stephenson, I973). However, the pronounced early peak, which in the K-BALB cells represents endogenous xenotropic virus production (Stephenson & Aaronson, I972; Aaronson & Stephenson, I973), is not seen with the revertant cells. This suggests that these revertant cells are not inducible for the xenotropic endogenous virus. Virus-related cell RNA was measured by hybridization of Ki-MSV-specific edna to an experimentally determined excess of cell RNA. Under these conditions, the concentration of virus-related nucleotide sequences is related to the rate of hybrid formation, as expressed by the Crt value (in tool. s/l) at which the reaction is half complete (Birnsteil et al. I972). This edna hybridizes nearly completely (approx. 96 %) to the homologous RNA. A lack

Short communications 247 a) (b) _ e- < Z, 40 60-40 60 100 10 ~ 10 4 lo s Cot (mol s/t) 100 lo 3 10 4 lo s Cot (tool s/l) Fig. 2. (a) Hybridization of Ki-MSV ~s[-7os RNA to infected cell DNA.

3 Short communications 247 a) (b) _ e- < Z, ~ 10 4 lo s Cot (mol s/t) 100 lo lo s Cot (tool s/l) Fig. 2. (a) Hybridization of Ki-MSV ~s[-7os RNA to infected cell DNA. 12SI-Ki-MSV 7oS RNA was hybridized to the indicated Cot as described in Methods. DNA was from: (3-0, NRK cells infected with and producing Ki-MSV-MuLV; 0-0, NRK cells nonproductively infected with Ki-MSV. (b) Loss of proviral DNA from K-BALB revertant cells. 125I-Ki-MSV 7oS RNA was hybridized to the indicated Cot as described in Methods. DNA was from: A-A, K-BALB cells; O-Q, A3I cells; ~,-A, K-BALB SR mz cells. of hybridization to RNA from avian myeloblastosis virus indicates that the cdna contains little poly(dt). Ki-MSV cdna was hybridized to a vast (Io -fold) weight excess of cytoplasmic RNA to the indicated Crt, as described above. K-BALB cytoplasmic RNA hybridized nearly as much of the input 3H-cDNA (approx. 8o %) as did virus RNA (Fig. I). In agreement with earlier results, the C,t½ of the hybridization reaction was similar for different RNA inputs and indicated a virus RNA content of approx, o.i %. In contrast, little (z to 5 %) of this cdna hybridized to the RNA from nontransformed BALB/3T3 cells. The final level of hybridization was not increased by increasing the RNA input. Hybridization to cytoplasmic RNA from the revertant cells resulted in low levels of hydridization (I5 %) similar to that seen in the nontransformed BALB/3T3 cells and did not increase with increasing levels of RNA. These results indicate that the reversion of these cells from the transformed parental phenotype is correlated with strongly reduced expression of Ki-MSV-related virus RNA within the cells. DNA from different cell lines was examined by hybridization in vast (> ~C-fold)weight excess to 125I-labelled Ki-MSV 7oS RNA to determine if the reversion to normal morphology and loss of expression of Ki-MSV-specific RNA was correlated with a loss or absence of Ki-MSV proviral sequences. DNA from NRK cells producing Ki-MSV was used as a positive control to test the maximum amount of hybridization of the 12SI-RNA. After hybridization to a Cot (uncorrected) of 8 x io 4, 45 % of the input RNA was hybridized. This amount was not increased at a Cot of I2 io 4 and therefore probably represents the maximum amount of RNA hybridizable under these conditions. All values were therefore normalized to I oo % for comparison. An essentially similar curve was generated by hybridization to DNA from NRK cells nonproductively infected with and transformed by Ki-MSV, thus confirming that the RNA primarily represents sequences of the transforming, replication-defective component of Ki-MSV. The Cot~ (uncorrected) for both reactions was about 3 IO z. The data are summarized in Fig. z(a). Hybridization of Ki-MSV RNA to DNA from K-BALB cells proceeded to a similar degree, but had a somewhat higher Crt½ (approx. 8 Io3), possibly indicating a lower concentration of Ki-MSV specific sequences than in the nonproducer NRK cells. The nontransformed parent BALB/3T 3 cells hybridized significantly less (6o %) of the Ki-MSV RNA. The hybridization to uninfected mouse cell DNA reflects the presence of sequences

248 Short communications within the sarcoma virus RNA which are shared by the Ki-MuLV helper virus and which are related to virus sequences endogenous to BALB/3T3 cells (Gelb et al.

4 248 Short communications within the sarcoma virus RNA which are shared by the Ki-MuLV helper virus and which are related to virus sequences endogenous to BALB/3T3 cells (Gelb et al. I97I; Viola & White, I973; Chattopadhyay et al. t974). DNA from the revertant cells contained somewhat less Ki-MSV-related sequences than did the original nontransformed BALB/3T3 parent line and at a somewhat lower concentration (Cot½ = 8 ro 3 compared to 4 to3) These data (shown in Fig. 2b) suggest that these phenotypically reverted cells have lost part or all of the exogenously added Ki-MSV genome and have possibly also lost some endogenous proviral sequences. This is consistent with the lack of inducibility of xenotropic virus production by these cells early after treatment with IdUrd. K-BALB cells are murine fibroblasts nonproductively infected with Ki-MSV. A previously described cell line derived from the K-BALB line as a single cell subclone, K-BALB SR 1212, has reverted to a normal phenotype. These revertant ceils lack a rescuable sarcoma virus. Results presented here show that part or all of the sarcoma provirus is absent from DNA of these cells. In this respect they resemble previously reported revertant clones of MSV-transformed nonproducer murine (Bensinger et al. I977) and feline (Frankel et al. I976) fibroblasts which lack rescuable sarcoma virus and some or all of the proviral DNA sequences, as well as Rous sarcoma virus transformed murine cells which lack rescuable sarcoma virus (Svoboda et al. I977). The correlation of loss of rescuable sarcoma virus, phenotypic reversion and loss of proviral DNA may thus be relatively common. Hybridization of Ki-MSV tracer RNA proceeds to a lesser extent and more slowly to revertant cell DNA than to DNA from normal, uninfected murine fibroblasts. This indicates that some of the proviral sequences for endogenous viruses in BALB/C cells, which are related to sequences in Ki-MSV, may also be absent. Consistent with this possibility is the lack of dexamethasone-stimulated virus production early after treatment of these cells with IdUrd. Since the revertant cell line in these studies has a lower median number of chromosomes than the transformed parent (Nomura et al. I974), it seems likely that proviral sequences could be lost in chromosomal rearrangements. The data, however, do not preclude the possibility that the provirus could be specifically excised by mechanisms similar to those in bacteriophage systems. Laboratory of Tumour Cell Biology National Cancer Institute National Institutes of Health Bethesda, Md. 014, U.S.A, C. D. TRAINOR M. S. REITZ, JUN. REFERENCES AARONSON, S. A. & STEPHE~SON, J. R. (1973). Independent segregation of loci for activation of biologically distinguishable RNA C-type viruses in mouse cells. Proceedings of the National Academy of Sciences of the United States of America 7o, AARONSON, S. A. & WEAVER, C. A. (I971). Characterization of murine sarcoma virus (Kirsten) transformation of mouse and human cells. Journal of General Virology x3, BENSINGER, W. I., ROBBINS, K. C., GREENBERGER, J. S. & AARONSON, S. A. 0977)- Different mechanisms for morphological reversion of a clonal population of murine sarcoma virus-transformed nonproducer cells. Virology 77, 75o-76I. BENVENISTE, R. E. & SCOLNICK, E. M. (1973)- RNA in mammalian sarcoma virus-transformed nortproducer cells homologous to murine leukemia virus RNA. Virology 5I, 37o-382. BIRNSTEIL, M. L., SELLS, 1~. n. & PURDOM, L F. (I972). Kinetic complexity of RNA molecules. Journal of Molecular Biology 63, CHATTOPADIqYAY, S., LOWY, D., TrICH, N., LEVtNr, A. & ROWE, W. (I974). Evidence that the AKR murine leukemia virus genome is complete in DNA of the high virus AKR mouse and incomplete in the DNA of the 'virus-negative' NIH mouse. Proceedings of the National Academy of Sciences of the United States of America 7I, I67-I7t.

Short communications 249 DUC-NGtlVEN, H., ROSENaAUM, E. N. & ZEIGEL, R. r. (I966). Persistent infection of a rat kidney cell line with Rauscher leukemia virus. Journal of Bacteriology 92, I 133-I t4o.

5 Short communications 249 DUC-NGtlVEN, H., ROSENaAUM, E. N. & ZEIGEL, R. r. (I966). Persistent infection of a rat kidney cell line with Rauscher leukemia virus. Journal of Bacteriology 92, I 133-I t4o. FRANKEL, A. E., HAAPALA, D. K., NEUBAUER, R. L. & FISCHINGER, P. J. (I 976). Elimination of the sarcoma genome from murine sarcoma virus-transformed cat cells. Science xgx, GELB, L., AARoNsoN, S. A. & MARTIN, M. 0971). Heterogeneity of murine leukemia virus in vitro DNA; detection of viral DNA in mammalian cells. Science x72, MAISEL, J., KLEMENT, J., LAI, M. M.-C., OSTERTAG, W. & DUESBERG, P. (I973). Ribonucleic acid components of murine sarcoma artd leukemia viruses. Proceedings of the National Academy of Sciences of the United States of America 7 o, o. NOMURA, S., DUNN, K. J., MATTERN, C. E. T., HARTLEY, J. W. & FISCHINGER, P. J. (I974). Revertants of mouse cells transformed by murine sarcoma virus: fiat variants without a rescuable sarcoma virus from a clone of BALB/3T 3 transformed by Kirsten MSV. Journal of General Virology 25, zo REITZ, M. S., MILLER, N. R., WONCJ-STAAL, F. W., GALLAGHER, R. E., GALLO, R. C. & GILLESPIE, D. 1-I. (1976). Primate type-c virus nucleic acid sequences (woolly monkey and baboon types) in tissues from a patient with acute myelogenous leukemia and in viruses isolated from cultured cells of the same patient. Proceedings of the National Academy of Sciences of the United States of America 73, REITZ, M. S., wtr, A. M. & GALLO, R. C. (I977). Synthesis of type-c virus particles from murine cultured cells induced by iododeoxyuridine. VL Biosynthesis of reverse transcriptase. International Journal of Cancer, STEPHENSON, J. R. & AARONSON, S. A. (1972). A genetic locus for inducibility of C-type virus in BALB/C cells: the effect of a nonlinked regulatory gene on detection of virus after chemical activation. Proceedings of the National Academy of Sciences of the United States of America 69, oi. STEPHENSON, J. R., REYNOLDS, R. K. & AARSONSON) S. A. (1973)- Characterization of morphologic revertants of murine and avian sarcoma virus-transformed cells. Journal of Virology xx, SVOBODA, J., POPOVIC, M., SAINEROVA, H., MACH, O., SHOYAB, M. & BALUDA, M. (I977). Incomplete viral genome in a non-virogenic mouse tumour cell line (RVPa) transformed by Prague strain of avian sarcoma virus. International Journal of Cancer x9, V1OLA, M. & WmXE, L. 0973). Differences in murine leukemia virus-specific DNA sequences in normal and malignant cells. Nature 246, WU, g. M., REITZ, M. S., PARAN, M. & GALLO, R. C. (I974). Mechanism of stimulation of murine type-c RNA tumour virus production by glucocorticoids: post-transcriptional effects. Journal of Virology 14, 8o w~, A. M., SCHULWZ, A. & GALLO, R. C. (1976). Synthesis of type-c virus particles from murine cultured cells induced by iododeoxyuridine. V. Effects of interferon and dexamethasone. Journal of Virology x9, Io8-I I7. (Received I6 October I978)

Separation of sarcoma virus-specific and leukemia virus-specific genetic sequences of Moloney sarcoma virus (mechanism of transformation)

Proc. Nat. Acad. Sd. USA Vol. 72, No. 11, pp. 4650-4654, November 1975 Microbiology Separation of sarcoma virus-specific and leukemia virus-specific genetic sequences of Moloney sarcoma virus (mechanism