of three morphologically different cell lines originating from. DBA/2J fetal liver cells transformed by the anemic strain of. Friend leukemia virus in vitro were ...
Proc. Nati. Acad. Sci. USA Vol. 77, No. 10, pp. 5769-5773, October 1980
Biochemistry
Variations in properties of virus released from morphologically different cell lines transformed in vitro by Friend leukemia virus (reverse transcriptase/viral RNA/gel electrophoresis)
D. TsUEI, B. G.-T. POGO, AND C. FRIEND Center for Experimental Cell Biology, Mollie B. Roth Laboratory, Mount Sinai School of Medicine, City University of New York, New York, New York 10029
Contributed by Charlotte Friend, July 8,1980
ABSTRACT The properties of the virus synthesized by each of three morphologically different cell lines originating from DBA/2J fetal liver cells transformed by the anemic strain of Friend leukemia virus in vitro were analyzed. The cells of line C-1 are malignant in syngeneic DBA/2 mice, grow in suspension, and are erythroid in origin. Cells of lines G-2 and G-3 are adherent, are epithelial in appearance, and produce no tumors in DBA/2J mice. Higher reverse transcriptase activity was detected in the culture fluid of lines G-2 and G-3, although virus from G-1 cells was more leukemogenic. Differences were also found in the virion density and size of the viral genome. RNA from the virions produced by G-2 and G-3 cells sedimented at 75 S in a sucrose gradient; virion RNA from C-1 cells sedimented at 60 S. However, when subjected to polyacrylamide gel electrophoresis, all three virus strains showed identical RNA subunits with an estimated molecular weight of 2.6 X 106. Analysis of virion proteins by slab gel electrophoresis showed differences in envelope protein (gp7l) components but not in the major core protein (p30). The properties of these viruses are stable and remain unchanged after passage in 3T3 cells.
The anemic strain of Friend leukemia virus (FLV-A) is a competent virus with the ability to induce erythroleukemia in the absence of spleen focus-forming virus (SFFV) (1). The primary target cell for FLV is most likely an erythroid precursor (2). That infection may also affect other cell types (3) was supported by the finding that permanent lines of morphologically. different cells originated from cultures of fetal liver transformed by FLV-A in vitro. Three of these lines have been characterized (4). The cells of line G-I are immature hematological cells and resemble the erythroleukemia lines established from the spleens of FLV-infected mice (5). They grow in suspension and can be induced to differentiate along the erythroid pathway. The cells of lines G-2 and G-3 are adherent and epithelial. All three lines are chronically infected with virus. In view of the findings that greater levels of virus were produced by the adherent lines and that this virus was less leukemogenic than that produced by the suspension lines, it was important to compare the molecular properties of the virus of each of the three transformed cell lines. The virus produced by the adherent lines was found to differ from that of the suspension line in the virion particle density, sedimentation coefficient of high molecular weight (HMW) RNA, and viral envelope protein components. However, all of the virus strains have identical RNA subunits. MATERIALS AND METHODS Cells and Viruses. Establishment, growth conditions, and characterization of the FLV-A-transformed lines of G-1, G-2, and G-3 (4) as well as of the prototype Friend erythroleukemia cell line 745A (5) and its subclone 5-86 (6, 7) have been de-
scribed. Generally, cells seeded at a concentration of 1 X 105 cells per ml were maintained in basal medium/Eagle (GIBCO) supplemented with 10% fetal calf serum, penicillin, and streptomycin. Cultures of 3T3 cells were infected 24 hr after seeding at 5 X 105 cells per 75-cm2 Falcon flask. They were treated with 2 ml of DEAE-dextran (25 Ag/ml) for 1 hr at 370C before 2.0 ml of the virus to be tested was added (8). After 1 hr at 370C to allow for virus adsorption, the cells were washed and incubated with fresh medium. Virus in the culture fluid was harvested for assay or further purification, as indicated in the text. Avian myeloblastosis virus and Rauscher leukemia virus were used as markers for the determination of the size of viral RNA or proteins. Virus Replication. Virus released into the culture fluid was assayed for syncytia formation on XC cells (9). Generally, leukemogenicity was assayed in 6- to 8-week-old DBA/2J mice inoculated intravenously with 0.5 ml of the filtered culture fluid. Reverse Transcriptase (RTase) Assay. The amount of virus released into the culture fluid was measured by incorporation of [3H]deoxythymidine monophosphate, using poly(rA.dT)lo as template-primer under the conditions described previously
(6).
Purification and Labeling of Virus. Medium from cultures that had not reached saturation density was harvested and mixed with 10% polyethylene glycol (Fisher) and 0.5 M NaCl to precipitate virus (10). After two successive 15-55% sucrose gradient centrifugations at 36,000 rpm for 1 hr in an SW 40 rotor, virus banding at density 1.16-1.18 g/cm3 was centrifuged under the same conditions and the pellet was resuspended in 0.1 M NaCl/10 mM Tris-HCl, pH 7.6/1 mM EDTA (NTE). Purified virus preparations were stored at -80'C before use. To prepare isotope-labeled virus, cells in the logarithmic phase were incubated for 17 hr with [5',6'-3H] uridine (41 Ci/ mmol; 1 Ci = 3.7 X 1010 becquerels; Schwarz/Mann) or32P04 (New England Nuclear) at 20 ,uCi/ml. The culture fluid was layered on a cushion of 1.0 ml of 25% sucrose in NTE and centrifuged at 36,000 rpm in a Spinco SW 40 rotor for 1 hr. The virus pellet was then resuspended in 0.3 ml of NTE and centrifuged on a 15-55% linear sucrose gradient at 45,000 rpm in a Spinco SW 65 rotor for 1.5 hr. The fraction containing the virus peak was collected and centrifuged again at 45,000 rpm for 1.5 hr in the SW 65 rotor. The pellet was resuspended in NTE and the viral RNA was extracted three times with phenol and NaDodSO4 (11). Abbreviations: FLV-A, anemic strain of Friend leukemia virus; FLV-P,
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polycythemic strain of Friend leukemia virus; SFFV, spleen focusforming virus; RTase, reverse transcriptase; NTE buffer, 0.1 M NaCI/10 mM Tris.HCI, pH 7.6/1 mM EDTA; HMW, high molecular weight; kDal, kilodalton. 5769
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Proc. Natl. Acad. Sci. USA 77 (1980)
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at 100 V for 3 hr. Gels were sliced into 2-mm fractions and digested with tissue solubilizer (NCS, Amersham) for 2 hr at 400C, and the radioactivity was determined. NaDodSO4/Polyacrylamide Gel Electrophoresis of Viral Protein. Purified virus preparations were resuspended in a buffer, heated at 1000C for 10 min in sample buffer containing 1% NaDodSO4, and electrophoresed on 10-20% polyacrylamide slab gels for 2-5 hr (13). Gels were stained with 0.25% Coomassie brilliant blue (Bio-Rad) in 50% methanol and destained with 10% acetic acid. Nuclease Assays. RNase Phy-1, RNase C, RNase H, and Escherichia cohl RNase III were assayed according to the conditions described (14-17), using RNA extracted from virus purified from 5-86 and G-2 cultures. All enzymes were purchased from Enzo Biochemicals (New York) except RNase H which was purchased from Miles.
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RESULTS Virus Activity. Exogenous RTase activity in the medium of each of the three FLV-A-transformed lines was compared to that of prototype clone 5-86 as a measure of virus release (Fig. 1). The virus released from G-1 cells had approximately the same level of RTase activity as that from line 5-86; however, the enzymatic activity in the same amount of culture fluid (50 gl) from G-2 or G-3 cultures was more than 10 times higher. Under the culture conditions used, the peak of accumulated RTase activity usually occurred about 3 days after seeding, when the cells were at the late logarithmic phase of growth. It is not likely that the differences in RTase activity are due to the variations in the saturation density of each of the lines. Although lines G-2 and 5-86 reached approximately the same final cell density, the level of RTase activity in the medium differed markedly. Similar differences in activity were also demonstrable when the virus was assayed on XC cells. Virus from lines 5-86 and G-1 caused few syncytia even when added without dilution to the cells, whereas virus from lines G-2 and G-3 formed syncytia up to a dilution of 1:100,000. However, viral activity, as estimated by the two In vitro assays did not correlate with the leukemogenicity. Virus from lines 5-86 and G-1 induced leukemia in about 10% of the inoculated animals. However, virus synthesized by the adherent lines was immunogenic. Vaccinated DBA/2 mice are solidly protected against challenge with the highly leukemogenic mouse-passaged
o
Time, hr FIG. 1. Accumulation of viral RTase activity. Aliquots from the tissue culture wvere harvested daily for RTase assay. Each point represents the mean of duplicate reaction mixtures after zero time -reaction had been subtracted; 1 pmol of [,3H]TMP incorporated is equivalent to 2000 cpm. 4, G-1; *, G-2; v, G-3;10 5-86.
Analysis of Virion RNA and Its Subunit. Sedimentation of [3H]uridine- or 32P-labeled viral HMW RNA was carried out in a 5-20% linear sucrose gradient centrifuged at 45,000 rpm for 1.5 hr in a Spinco SW 65 rotor. The gradients were fractionated by collecting 10 drops on filter discs (Whatman 3 MM filter) and washed with trichloroacetic acid and ethanol (12). For the studies on the subunit size, [3H]uridine-labeled 5-86 virus RNA and 32P-labeled G-1, G-2, or-G-3 virus RNA were mixed and denatured at 70'C in 4 M urea for 3 min. The mixture was then electrophoresed on a 2% polyacrylamide gel
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30 0 10 20 20 30 Fraction FIG. 2. Sedimentation profiles of virions from FL cells in sucrose gradients. 32P-Labeled virus (0) purified from medium of lines G-1 (Left), G-2,(Middle), or G-3 (Right) was mixed with [3H]uridine-labeled virions (-) from line 5-86 and sedimented in a 15-55% sucrose gradient at 45,000 rpm for 90 min in a Spinco SW 65 rotor. 0
20
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Proc. Natl. Acad. Sci. USA 77 (1980)
Tsuei et al.
Biochemistry:
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FLV-A. There was a difference of 3 orders of magnitude in the median lethal dose (LD5o) between control and immunized mice (data not shown). Sedimentation Profiles of the Virion and its RNA. The sedimentation profiles of the virus released by the different cell lines are compared in Fig. 2. Virus particles from each line were pulse-labeled with 32P and cosedimented with [3H]uridinelabeled virions from line 5-86. Virions from lines G-1 and 5-86 sedimented at a density of 1.16-1.17 g/cm3. Virions from line G-2 or G-3 were slightly heavier and sedimented at a density of 1.17-1.18 g/cm3. The virion RNA from these lines was also studied. Pulselabeled RNA from purified virions of each of the three lines was cosedimented with 5-86 virion RNA in a linear sucrose gradient (Fig. 3). The RNA from the virions of line 5-86, which sediments at 68 S as compared to avian myeloblastosis virus RNA, was used as a marker. The virion RNA of line C-1 had the same sedimentation coefficient as that of the 5-86 virion. The RNA from the virions of lines G-2 and G-3 sedimented at 75 S, about 25% heavier. The size of the RNA subunit from virions of each cell line was 28S
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Fraction FIG. 4. Polyacrylamide gel electrophoresis of heat-dissociated RNA of virus from line 5-86. Viral RNA (O 0) was heated in 4 M urea for 4 min at 70°C and then coelectrophoresed with ribosomal RNA (0--). ---
also examined. 32P-Labeled RNA extracted from the purified virions of line 5-86 was denatured in 4 M urea and coelectrophoresed with marker 28 S and 18 S ribosomal RNA (rRNA) on the gels (Fig. 4). From its position slightly behind 28S rRNA, the molecular weight of the subunit was estimated to be about 2.6 X 106. The RNA subunit of 5-86 virions was then coelectrophoresed with RNA subunits of G-1, G-2, or G-3 virions. The mobilities of the subunits of all four strains of virus were iden-
tical, indicating that their size was the same (Fig. 5). Sensitivity to Nucleases. To determine whether there was a major change in the structure of the RNAs extracted from the virions of the G-1 and G-2 lines, the sensitivity of the RNAs to nucleases was studied. Both RNAs were insensitive to digestion with RNase H and E. colh RNase III, indicating that neither RNA-DNA hybrids or double-stranded RNA was present. In addition, there was no difference in the rate of hydrolysis of these RNAs when they were exposed to RNase C or RNase Phy-1. RNase C preferentially attacks C residues and RNase Phy-I attacks G, U, and A residues (data not shown). Analysis of Virion Structural Proteins. The structural proteins of the virions from each of the cell lines were analyzed by NaDodSO4/polyacrylamide gel electrophoresis. The 30kilodalton (kDal) core proteins of the four virus strains were identical, but slight differences in the mobility of 12- to 15-kDal component of G-2 and G-3 virions were observed (Fig. 6). The major envelope protein components of the virus also showed variations. These major glycoproteins from G-1 virions and from 5-86 virions were 60 kDal, compared to those of G-2 and G-3 virions which were in the range of 68-72 kDal. However, one or two minor bands in the range of 60-72 kDal were also observed in these virus strains. Virus Released from Infected 3T3 Cells. In order to determine whether the host cell contributed to differences in the properties of the virus, 3T3 cells were infected with 105 XC plaque-forming units of G-2 or G-3 virus per ml. The virus produced had the same level of RTase and XC leukemogenic activities as the virus before growth in 3T3 cells (data not shown). The sedimentation density of the passaged virus was the same as for the G-2 or G-3 virion, 1.17-1.18 g/cm3. There was no difference in the sedimentation rate of the RNAs or in the viral protein components from virus before and after passage through 3T3 cells. However, it was not possible to assay the progeny virus from 3T3 cells infected with G-1 or 5-86 virus before four to six passages, possibly due to the low level of the virus used for infection. Assays done 3 weeks after the initiation
5772
Biochemistry:
Proc. Natl. Acad. Sci. USA 77(1980)
Tsuei et al.
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40 20 Fraction FIG. 5. Polyacrylamide gel electrophoresis of heat-dissociated RNA (0) of virus from lines G-1 (Left), G-2 (Middle), and G-3 (Right) and from line 5-86 (0).
of the 3T3 cultures indicated that the sedimentation of virus and RNA was unchanged after passage (data not shown).
DISCUSSION The finding that viral RTase activity in the culture fluid of the FLV-A-transformed adherent lines C-2 and GC3 was consistently higher than that detected in the culture fluid of the suspension line GC- prompted us to compare the molecular properties of the virus from each of the lines. Differences were found in the virion density and the size of the viral genome. The sedimentation coefficient of HMW RNA from the virions of lines G-2 and C-S was estimated to be approximately 75 S as compared to 60 S for virions from G-1 and 5-86 cells (Fig. 3). --~~~~~
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FIG. 6. NaDodSO4/polyacrylamide slab gel electrophoresis of viral proteins. Size markers (Bio-Rad) are shown: actin, 45 kDal; bovine serum albumin, 68 kDal; and rabbit muscle actin, 100 kDal.
The possibility that the high sedimentation coefficient of the virion RNA of lines C-2 and G-3 may be due to a difference in configuration, hydrodynamic properties, host cell modification, or contamination with a tightly bound protein of the HMW RNAs is not yet ruled out. Our value for the sedimentation coefficient of virion RNA of line 5-86 is compatible with that of Maisel et al. (18) for another Friend erythroleukemia cell line. However, the subunit RNA from each of these virus strains has identical mobility when subjected to gel electrophoresis, and has anestimated Mr of 2.6 X 106 (Fig. 4). This is in line with the report that Rous sarcoma virus contains a population of HMW RNAs that have indistinguishable electrophoretic mobilities although they have heterogeneous sedimentation coefficients (19). It has also been noted that the sedimentation coefficient of RNA may increase with the maturation time of the virus (20) as well as with increased concentration of Mg2+
(19).
No major structural differences between the viral RNAs derived from the suspension and the adherent lines were detected. The results of experiments using different nucleases indicate that no double-stranded nucleic acids were present. In addition, no differences in the rate of hydrolysis of the RNAs occurred when these RNAs were exposed to RNase C or RNase Phy-1. Each of the three FLV-A-transformed lines yielded virus particles that sedimented at a single density characteristic for the strain and had only one component in the HMW RNA sedimentation profile (Figs. 2 and 3). However, it has been reported that the polycythemic strains of the virus, which are a complex of FLV and SFFV (21), sediment at two densities. A bouyant density of 1.14 g/cm3 distinguishes the SFFV component; the leukemia virus component sediments at a bouyant density of 1.16-1.17 g/cm3 (22). Particles with density 1.14 g/cm3 were not detected in virus preparations from the FLV-A-transformed cell lines described here and the Mr of the RNA subunits of these viruses (2.6 X 106) is equivalent to the medium size of the three species of subunits (35 S, 32 S, and 30 S) in the virus released from Friend erythroleukemia cell lines developed by Ostertag et al. (11, 23). The differences between our results and theirs may be a reflection of the virus strains used in developing the cell lines. The lines used by those investigators originated from transformed cells in the leukemic spleens of mice infected with FLV-P strains of the virus (24), and the lines in the present study originate from cells transformed in vitro with the FLV-A strain which does not appear to contain this SFFV component. Analysis of the virion proteins by slab gel electrophoresis also shows that there are differences between the envelope proteins,
Biochemistry:
Tsuei et al.
ranging from 60 to 72 kDal, but not in the major core proteins (30 kDal) (Fig. 6). It is not known whether the variations in the envelope proteins are due to glycosylation or to structural differences (25). In order to determine whether the size of the genome and the pattern of the proteins are stable features and not a reflection of host cell modification, each virus was passaged in 3T3 cells. However, thus far no significant changes in biological and biochemical properties have been observed. Thus, it is possible that the variations in the viral properties may have derived from the selection of transformed cells from a heterogeneous population of infected liver cells. Although it is not known whether there was host cell modification at the time of the initiation of infection, it seems, at least from the observations on virus passaged in 3T3 cells, that the viral properties remained stable and were not altered by the host cell in this case. Further studies on the nucleic acid sequence homology, oligonucleotide fingerprinting, and tryptic mapping of viral proteins of these virus strains may reveal the source of the variability of the virions. We thank Dr. Yair Gazitt for asssance in the slab gel electrophoresis and Mr. J. Gilbert Holland and Paul Freimuth for expert technical assistance. We also thank Dr. Claudio Basilico for the 3T3 cells, Dr. Joseph Beard for avian myeloblastosis virus, and the Office of Program Resources and Logistics (National Cancer Institute) for the Rauscher leukemia virus. This work was supported in part by National Institutes
of Health Grants CA 10,000, CA 13,047, and AI 15953. 1. Oliff, A. I., Hager, G. L., Chang, E. H., Scolnick, E. M., Chan, H. W. & Lowy, D. R. (1980) J. Virol. 33,475-486. 2. Tambourin, P. E. & Wendling, F. (1975) Nature (London) 256, 320-322. 3. Golde, D. W., Faille, A., Sullivan, A. & Friend, C. (1976) Cancer Res. 36, 115-119. 4. Golde, D. W., Bersch, N., Friend, C., Tsuei, D. & Marovitz, W. (1979) Proc. Natl. Acad. Sci. USA 76,962-966.
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5. Friend, C., Preisler, H. D. & Scher, W. (1974) in Current Topics in Developmental Biology, eds. Monroy, A. & Moscona, A. A. (Academic, New York), pp. 81-101. 6. Scher, W., Tsuei, D., Sassa, S., Price, P., Gabelman, N. & Friend, C. (1978) Proc. Natl. Acad. Sci. USA 75,3851-3855. 7. Tsuei, D., Haubenstock, Revoltella, R. & Friend, C. (1979) J. Virol. 31, 178-183. 8. Toyoshima, K. & Vogt, P. K. (1969) Virology 38,414-426. 9. Rowe, W. P., Pugh, W. E. & Hartley, J. W. (1969) Virology 42, 1136-1139. 10. Green, M., Rokutanda, M., Fujinaga, K., Ray, R. K., Rokutanda, H. & Gurgo, C. (1979) Proc. Natl. Acad. Sci. USA 67, 385393. 11. Dube, S., Kung, H.-J., Bender, W., Davidson, N. & Ostertag, W. (1976) J. Virol. 20, 264-272. 12. Scher, W. & Friend, C. (1978) Cancer Res. 38, 841-849. 13. Laemmli, V. K. (1970) Nature (London) 227,680-685. 14. Simoncsits, A., Brownlee, G. G., Brown, R. S., Rubin, J. R. & Guilley, H. (1977) Nature (London) 269,833-836. 15. Schmukler, M., Jewett, M. & Levy, C. C. (1975) J. Biol. Chem. 250,2206-2212. 16. Berkower, I., Leis, J. & Hurwitz, J. (1973) J. Biol. Chem. 248, 5914-5931. 17. Robertson, H. D., Webster, R. E. & Zinder, N. D. (1968) J. Biol. Chem. 243, 82-91. 18. Maisel, J., Klement, V., Lai, M. M.-C., Ostertag, W. & Duesberg, P. (1973) Proc. Natl. Acad. Sci. USA 70,3536-3540. 19. King, A. M. Q. (1976) J. Biol. Chem. 251, 141-149. 20. Cheung, K.-S., Smith, R. E., Stone, M. P. & Joklik, W. K. (1972) Virology 50, 851-864. 21. Steeves, R. A., Eckner, R. J., Bennett, M., Mirand, E. A. & Trudel, P. J. (1971) J. Natl. Cancer Inst. 46, 1209-1217. 22. Eckner, R. J. & Hettrick, K. L. (1977) J. Virol. 24, 383-396. 23. Ostertag, W. & Pragnell, I. (1978) Proc. Natl. Acad. Sci. USA 75, 3278-3282. 24. Ostertag, W., Melderis, H., Steinheider, G., Kluge, N. & Dube, S. (1972) Nature (London) New Biol. 239,231-234. 25. Bolognesi, D. P., Montelaro, R. C., Frank, H. & Schafer, W. (1978) Science 199, 183-187.
