VIROLOGY
90,
Murine
AKHIL
12-22
(1978)
Mammary Tumor Virus (MuMTV) Infection of an Epithelial Line Established from C57BL/6 Mouse Mammary Glands B. VAIDYA,’
ETIENNE Y. LASFARGUES, WILLIAM G. COUTINHO
Department of Microbiology and Immunology, The Hahnemann Pennsylvania 19102 and ins&&e for Medical Research, Accepted
June
JOEL
Medical Camden,
B. SHEFFIELD,’
Cell
AND
College, Philadelphia, h&w Jersey 08103
5, 1978
An epithelioid cell line derived from the mammary glands of a C57BL/6 mouse and designated C57MG cell line was found to be susceptible to infection by murine mammary tumor virus (MuMTV). Although uninfected C57MG cells contain endogenous MuMTVrelated DNA sequences, no RNA sequences homologous to MuMTV were detectable, even after treatment with the glucocorticoid, dexamethasone. However, after infection with MuMTV, these cells acquire additional MuMTV DNA, and viral RNA and proteins were readily detectable. Most, if not all, of the additional MuMTV DNA in infected C57MG cells appeared to be integrated. Synthesis of viral RNA and protein, and the release of virions into culture fluid by infected C57MG cells, was stimulated by incorporation of dexamethasone in the growth medium. The efficiency of infection by MuMTV in C57MG cells was similar to that in nonmurine cells. There was a direct relationship between multiplicity of infection (m.o.i.) and the average number of MuMTV RNA molecules detected per cell 5 weeks after infection. However, even at the highest m.o.i. used (4 X lo” virions/cell), a plateau in the average number of viral RNA molecules per cell was not achieved. The origin of MuMTV synthesized by infected C57MG cells, whether it is the progeny of infecting MuMTV or of the endogenous C57BL/6 MuMTV or of both, remains undetermined. We have been unable to detect anv mornholo~icai or growth pattern changes in C57MG cells following infection with MuM’k’. -
(Ringold et al., 1977a) and Howard et al. (1977) have confirmed the susceptibility of cat kidney and mink lung cells to infection by MuMTV. Several interesting features have emerged from MuMTV infection of nonmurine cells, which include the following. (1) The amount of MuMTV RNA present in nonmurine cells is also stimulated by glucocorticoids (Vaidya et al., 1976; Ringold et al., 1977a), just as it is in cultures derived from mouse mammary tumors that synthesize MuMTV (Ringold et al., 1975; Parks et al., 1975). This would indicate that either MuMTV proviral DNA integrates in nonmurine cells at a site adjacent to the sequences that are recognized by the glucocorticoid receptor complex or that MuMTV proviral DNA itself carries such recognition sequences. (2) When stimulated with glucocorticoids, infected nonmurine cells ac-
INTRODUCTION
A detailed analysis of the biology of murine mammary tumor virus (MuMTV), the only retravirus known whose main pathological manifestation is a hormonally controlled carcinoma, has been hampered for many years due to the lack of appropriate in vitro systems. Until recently, a major block had been the inability to infect cultured cells with MuMTV. We have reported previously that epithelioid cell lines derived from cat kidney and mink lung are susceptible to infection by MuMTV (Lasfargues et al., 1974, 1976a, b; Vaidya et al., 1976). This observation was later extended to a cell line derived from a rat hepatoma ’ To whom reprint requests should be addressed at the Department of Microbiology and Immunology, the Hahnemann Medical College, Philadelphia, Pa. 19102. ’ Present address: Department of Biology, Temple University, Philadelphia, Pa. 19122. 12 0042-6822/78/0901-0012$02.00/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
MuMTV
INFECTION
cumulate unintegrated MuMTV DNA molecules in linear as well as in covalently closed circular duplex forms (Ringold et al., 197713; Vaidya, unpublished observations). Such molecules have been used in generating a restriction endonuclease cleavage map of MuMTV (Shank et aZ., 1978). And, (3) infection of nomnurine cells has provided progeny virions for use in comparative studies of viral proteins obtained from different strains of MuMTV so as to rule out host-mediated modification (Teramoto et al., 1977). However, appropriate biological studies on MuMTV can best be carried out when its target cell, i.e., the mammary epithelium of mouse, is infected with the virus. In this paper, we describe MuMTV infection of an epithelioid cell line established from the mammary glands of a C57BL/6 mouse. A preliminary report of these results was presented at the 68th Annual Meeting of the American Association for Cancer Research in Denver, Colorado (Vaidya and Lasfargues, 1977). MATERIALS
AND
METHODS
Cells and virus. A cell line, designated C57MG, was established by one of us (W.G.C.) from the mammary glands of a 23-week-old C57BL/6 female mouse. The mammary glands were dissociated overnight with 200 units/ml of collagenase (Worthington) and epithelial cells were separated on a O-50% Ficoll (Pharmacia) gradient by centrifugation at 8000 g for 1 hr. The cells were epithelial in morphology and were grown in Hanks’-Eagle’s Minimal Essential Medium (HE-MEM) containing 10 pg/ml insulin and 10% fetal bovine serum. MuMTV was purified from the milk of the RI11 strain of mice by sucrose density gradient. For the preparation of inoculum, purified virions were resuspended in HEMEM and passed through a 0.45~pm membrane filter that was pretreated with a 1% solution of polyvinylpyrrolidone-K-90 (GAF Corp.). The concentration of virions was determined by molecular hybridization assay as described below. Infection with MuMTV. Infection of C57MG cells was carried out using the techniques described previously for nonmurine
OF
MOUSE
CELLS
13
cells (Lasfargues et al., 1976; Vaidya et al., 1976). Briefly, about lo6 cells were resuspended per ml of HE-MEM containing 4 pg/ml polybrene (Aldrich) and about 10” MuMTV particles per ml. After 1 hr at 37” with intermittent agitation, the cell-virus suspension was diluted with complete medium and plated on culture flasks. For the experiments involving titration of MuMTV, constant numbers of cells were infected with logarithmic dilutions of MuMTV. Extraction of nucleic acids. Cultured cells were lysed in extraction buffer (0.05 M Tris-HCl, pH 8.0, 0.02 M EDTA, 0.1 M NaCl, and 0.5% sodium dodecyl sulfate) at a concentration of about lo7 cells/ml and treated with 200 pg/ml of proteinase K (EM Laboratories) at 37” for 90 min. After two extractions with buffer-saturated phenol, nucleic acids were precipitated with ethanol and stored at -20”. To purify DNA, nucleic acids were resuspended in 3 mM EDTA, pH 7.0, and adjusted to 0.3 M NaOH. Following incubation in a boiling water bath for 20 min and neutralization with HCl, DNA was precipitated with cold ethanol. The alkali treatment hydrolyzed RNA and reduced the size of DNA chains to 300 to 400 nucleotides long (Tibbetts et al., 1973). To purify RNA, nucleic acids were resuspended in DNase buffer (0.02 M Tris-HCl, pH 7.4 and 0.01 M MgC12) and treated with 20 yg/ml of iodoacetate-treated (Zimmerman and Sandeen, 1966) RNase-free deoxyribonuclease I (Worthington) for 2 hr at room temperature. The solution was adjusted to 0.05 M Tris-HCl, pH 8.0, and 0.01 M EDTA, and then extracted twice with buffer-saturated phenol and precipitated with ethanol. Both DNA and RNA were resuspended at a concentration of >5 mg/ml in 3 mJ4 EDTA or 0.02 M Tris-HCl, pH 7.4, respectively. Separation of high molecular weight cellular DNA from low molecular weight unintegrated DNA was carried out according to the procedure of Hirt (1967). The cells were grown in roller bottles and lysed in situ with a solution containing 0.02 M TrisHCl, pH 7.5, 0.01 M EDTA and 0.6% SDS at a concentration of 0.5-l X lo7 cells/ml. After incubation on a roller apparatus for 5-10 min, the lysate was gently scraped off
14
VAIDYA
and made 1 M in NaCl by slow addition of 5 M NaCl with gentle mixing. The lysate was stored at 4’ overnight and the coagulate of high molecular weight DNA, protein and SDS was pelleted out by centrifugation at 15,000 g for 30 min. The pellet and the supernatants were separated and DNA was extracted as described above. RNA was extracted from lactating mammary glands of mice by homogenization of the glands in a Virtis homogenizer, followed by phenol extraction as described earlier (Vaidya et al., 1974). MuMTV cDNA. Highly radioactive DNA complementary to MuMTV RNA was synthesized by detergent-disrupted purified virions utilizing the endogenous RNA-directed DNA polymerase in the presence of actinomycin D as described before (Vaidya et al., 1974), and purified by batch elution from hydroxylapatite (BioRad). The specific activity of the cDNA was about 2 X lo7 cpm/pg. All the different batches of cDNA synthesized were specific and gave consistent results as judged by cDNA MuMTV 70 S RNA hybridization kinetics. Although a large proportion of the cDNA may be a multiple transcript of a small segment of the viral genome, we have found that when hybridization was conducted at a cDNA to MuMTV RNA ratio of 4, about 40% of the RNA was protected from RNase digestion under high salt conditions. The cDNAs synthesized by us have served as sensitive and accurate probes for the detection and quantitation of MuMTV nucleic acids. Molecular hybridization. Cellular DNA to MuMTV cDNA annealings were performed in 50 ~1 volume in conical polypropylene tubes. Various amounts of cellular DNA were mixed with about 1000 cpm of [3H]cDNA and adjusted to 0.6 M NaCl, 0.04 M Tris HCl, pH 7.2, and 0.002 M EDTA by addition of a concentrated buffer salt solution. The annealing mixtures were adjusted to a constant DNA content by addition of sheared salmon sperm DNA and covered with mineral oil. After incubation at 100’ for 10 min and quick chilling on ice, annealing was conducted at 68” for 66 hr. The extent of annealing was determined by resistance to single strand-specific nuclease
ET AL.
S1 as described previously (Vaidya et al., 1976). When using the low molecular weight DNA associated with Hirt supernatants, volumes that were equivalent to the volumes used for Hirt pellet DNA were employed in the annealing reactions. Hybridization of RNA to MuMTV cDNA was carried out in a lo-y1 volume as described (Vaidya et al., 1974, 1976). The average number of MuMTV RNA molecules per cell was estimated by comparing Crtljs (C,t at which half-maximum hybridization occurs) for the sample RNA with C,.tl,z for MuMTV 70 S RNA. Quantitation of MuMTV by molecular hybridization assay. The number of MuMTV particles in a sample was measured by molecular hybridization assay as described (Ringold et al., 1975; Vaidya et al., 1976). After pelleting the virions by centrifugation, RNA was purified by proteinase-K treatment and phenol extractions followed by ethanol precipitation in the presence of yeast RNA as a carrier and hybridized with MuMTV cDNA. The kinetics of hybridization was plotted as a function of VOt (the product of volume of sample in milliliters and time of incubation in hours). The concentration of MuMTV RNA was determined by obtaining Votl,z value ( Vat at which half-maximum hybridization occurred) and the virion content of the sample was estimated on the assumption that each mature MuMTV particle contains one 70 S RNA molecule. Thermal denaturation of hybrids. Approximately 10,000 cpm of MuMTV cDNA were hybridized to saturation with RNA from various sources. The hybrids were ethanol-precipitated and resuspended in 0.02 M Tris. HCl, pH 7.4. Aliquots of the hybrids were incubated at various temperatures for 10 min and the extent of denaturation was measured by digestion with S1 nuclease. Radioimmunoassay (RIA). The procedure for RIA detection and quantitation of gp55, the major glycoprotein of MuMTV, has been described elsewhere (Sheffield et al., 1977). Briefly, a cytosol preparation was made by disrupting the cells in a hypotonic buffer containing nonionic detergent NP-40 followed by a low-speed centrifugation to
MuM’I’V
15
1Nl”r;C;‘l’lUN
remove nuclei. The protein concentration of cytosols was determined by the method of Lowry et al. (1951) and the ability of this preparation to inhibit immunoprecipitation of ’ 51-labeled gp55 under limiting antibody concentration was measured. The concentration of gp55-related antigen was estimated by comparison with the immunoprecipitation inhibition curve generated by known quantities of unlabeled gp55. Electron microscopy. Cells growing in monolayers were fixed with 2.5% glutaraldehyde (pH 6.8) for 2 hr and rinsed with H20 overnight. After dehydration with graded ethanol, the monolayer of cells was removed by ethylene dichloride, washed with ethanol, and embedded in Epon-Ardldite mixture. Ultra-thin sections were cut with glass knives, mounted on copper grids, and stained with uranyl acetate and lead citrate. The sections were examined and photographed in a JEM-1OOB electron microscope.
RESULTS
C57MG
Cell Line
Mammary glands of mice contain three different types of epithelial cells, viz., alveolar, ductal, and myoepithelial. As shown by Slemmer (1974), these cell types have distinct heritable behavior and, in normal mammary glands, they do not seem to differentiate from a single progenitor stem cell The C57MG cell line was derived from the mammary glands of a 23-week-old retired C57BL/6 breeder. As shown in Fig. 1, the cells were of epithelial morphology, the origin of which was not determined. However, it is quite likely that the epithelial cells of C57MG cell line are either cells of alveolar, ductal, or myoepithelial origin, or a mixture of two or more types. The cells used for experiments described in this paper were uncloned; therefore, we cannot rule out the possibility that we were dealing with a mixed population of epithelial cells.
FIG. 1. Photomicrographs of C57MG cells growing in a monolayer. A, the cells at confluency showing epithelial morphology. B, a higher magnification micrograph of cells that have not reached confluency. Note the spreading on the substratum, which is typical of epithelial cells.
16
VAIDYA
After infection with MuMTV, we did not observe any morphological or growth characteristic changes in C57MG cells. In fact, uninfected and chronically infected C57MG cells were distinguishable only by means of MuMTV production by the infected cells as described below. Increase in MuMTV Prouiral Following Infection
Sequences
It has been recognized for some time that all cells of Mus musculus contain multiple of DNA sequences related to copies MuMTV (Varmus et al., 1972; Michelides and Schlom, 1975), although there may be some quantitative (Morris et al., 1977) or qualitative (Drohan et al., 1977) differences among different strains or cells from normal organs and mammary tumors. As shown in Fig. 2, C57MG cells also contain DNA sequences related to MuMTV as determined by annealing experiments using MuMTV cDNA. After infection with MuMTV, C57MG cells acquired additional MuMTV DNA sequences (Fig. 2). In this experiment, increasing amounts of DNA from C57MG control or infected cells were annealed to MuMTV cDNA in 50 ~1 volume for 66 hr. The total DNA content of the annealing reaction was kept constant (as a viscosity
Ld -
l l /
FIG. 2. Acquisition of additional MuMTV proviral DNA by infected C57MG cells. C57MG cells were infected with MuMTV at a high multiplicity and DNA was extracted from uninfected and chronically infected C57MG cells. Various amounts of cellular DNA were annealed with MuMTV cDNA, keeping the reaction volume and the time of incubation constant. The extent of annealing was determined by resistance of cDNA to S1 nuclease. (*). DNA from uninfected cells and (O), DNA from chronically infected cells.
ET
AL.
control) by adjustment with salmon sperm DNA. Fig. 2 shows that about 64% of MuMTV cDNA annealed to infected C57MG cellular DNA at the plateau of the curve as compared to about 45% annealed to uninfected C57MG DNA. In these experiments the driver DNA molecules, at the highest concentration, were only in moderate excess, and the differences in plateau levels observed for cDNA annealing to infected and uninfected C57MG DNA were perhaps due to the difference in driver DNA to cDNA ratios (Muto, 1977). About 10 pg of DNA from infected cells was sufficient to achieve half-maximum annealing with MuMTV cDNA under these conditions. In contrast, half-maximum annealing with C57MG DNA from uninfected cells required about 30 pg of DNA. Therefore, we estimate that the infection with MuMTV resulted in about a three-fold increase in MuMTV-related DNA sequences in the C57MG cells. In order to determine whether the additional MuMTV proviral DNA was integrated into the cellular DNA, we carried out extraction of cellular DNA according to the procedure of Hirt (1967). This procedure separates high molecular weight cellular DNA from unintegrated, relatively low molecular weight DNA. The cells were grown in the presence of lo-” M dexamethasone for 48 l-n-s before extraction. The hybridization procedure was similar to the one described for Fig. 2. After the Hirt extraction and purification, increasing amounts of high molecular weight DNA (Hirt pellet DNA) were annealed to MuMTV cDNA in a constant volume and identical conditions were used for the equivalent amounts (as per volume) of low molecular weight DNA (Hirt supernatant DNA). Figure 3 shows that Hirt pellet DNA from both uninfected and infected C57MG cells annealed to MuMTV cDNA to the extent shown in Fig. 2; that is, there was about a 3-fold increase in high molecular weight-associated MuMTV DNA after infection of C57MG cells with MuMTV. Hirt supernatant DNA showed a limited extent of annealing with MuMTV cDNA, but this was probably due to a low-level contamination of Hirt supernatant DNA with high molecular weight
MuMTV
1
10 iG HlRT PELLET
INFECTION
100
DNA OR ITS EWYRLENT
HlR,
SUPERHATIWT
FIG. 3. Most, if not all, of the additional MuMTV proviral DNA in infected C57MG cells is associated with high molecular weight cellular DNA. The procedure of Hirt (1967) was used to separate high molecular weight cellular DNA (Hirt pellet DNA) from low molecular weight unintegrated DNA (Hirt supernatant DNA) from both uninfected and chronically infected C57MG cells grown in the presence of IO-” it4 dexamethasone. Annealing with MuMTV cDNA was performed using various amounts of Hirt pellet DNA or its equivalent volume of Hirt supernatant DNA as described in Materials and Methods. Symbols: 0, 0, Hirt pellet and Hirt supernatant DNA, respectively, from chronically infected C57MG cells; +, 0 Hirt pellet and Hirt supernatant DNA, respectively, from uninfected C57MG cells.
DNA. Although these results are indicative of integrated MuMTV DNA in infected C57MG cells, covalent linkage of viral DNA with the host genome is not rigorously demonstrated. However, it does seem that most, if not all, of the increased MuMTV DNA sequences in infected C57MG cells are associated with high molecular weight DNA of the host. In similar experiments, it was determined that in MuMTV-infected nonmurine cells like rat hepatoma cells (Ringold et al., 1977b), cat kidney and mink lung cells (Vaidya, unpublished observations), some of the proviral sequences were present in unintegrated forms, in addition to the integrated MuMTV provirus. This is comparable to the avian system in which chicken cells infected with avian sarcoma virus (ASV) do not carry unintegrated provirus, but ASV-infected duck and quail cells do (Guntaka et al., 1976). MuMTV Synthesis Cells
by Infected
C57MG
No detectable MuMTV RNA synthesis is observed in uninfected C57MG cells even after treatment with dexamethasone. This
OF
MOUSE
CELLS
17
is in contrast to the finding of Varmus et al. (1973) and our own observation that lactating mammary glands of C57BL mice contain about 40 copies of MuMTV RNA per cell, even though no viral antigens or virions are produced. The reason for the lack of MuMTV RNA in C57MG cells may be that, since the cell line was established from the mammary glands of a nonlactating mouse, no transcription of the MuMTV genes may occur under these physiologic conditions. In contrast, C57MG cells infected with MuMTV synthesized MuMTV RNA; when not stimulated with dexamethasone, about 150 copies of MuMTV RNA were present per cell, on an average (Fig. 4). Inclusion of dexamethasone in the culture medium enhanced the concentration of MuMTV RNA lo-fold (Fig. 4). The fact that these MuMTV specific RNA molecules were engaged in viral protein synthesis was demonstrated by measuring the amount of the major viral glycoprotein, gp55, in these cells by radioimmunoassay. Figure 5 shows that, whereas uninfected C57MG cells were not synthesizing any detectable gp55-related antigens whether treated with dexamethasone or
FIG. 4. Intracellular concentration of MuMTV RNA in infected C57MG cells in the presence or absence of dexamethasone. About 10” C57MG cells infected with MuMTV were treated with 1O-5 M dexamethasone for 48 hr and total cellular RNA was purified from both treated and untreated infected C57MG cells. Kinetics of cellular RNA to MuMTV cDNA hybridization were performed as described in Materials and Methods. Intracellular concentration of MuMTV RNA in infected C57MG cells increased about lo-fold in cells treated with dexamethasone.
18
VAIDYA
ut
FIG. 5. Radioimmunoassay
COMPETING
ET
AL.
PROTEIN
(RIA) for the major glycoprotein of MuMTV, gp55, in infected and uninfected C57MG cells. The cytoplasmic protein was obtained from infected and uninfected C57MG cells that were grown in the absence (open symbols) or presence (closed symbols) of 10 ’ M dexamethasone for 48 hr. The ability of various amounts of the protein to inhibit precipitation of ““I-labeled gp55 by limiting concentration of anti-gp55 was measured as described in Materials and Methods. Symbols: +, 0, protein from infected C57MG cells; 0, 0 protein from uninfected C57MG cells.
FIG. 6. Release of MuMTV by infected C57MG cells grown in the presence of the absence of dexamethasone. Confluent cells were either left untreated or exposed to lo-” M dexamethasone for 48 hr after which the virions present in a known volume of the culture fluids were recovered by centrifugation. The RNA was purified and resuspended in a small volume of 0.02 M Tris. HCl, pH 7.4. Various dilutions of this RNA were hybridized in a lo-p1 volume with MuMTV cDNA for 20 hr and the extent of hybridization assayed by S1 nuclease. The percentage hybridization was plotted as a function Vd as described in Materials and Methods. Symbols: 0, cells grown in the absence of dexamethasone; +, growth in the presence of dexamethasone.
not, the infected cells when unstimulated contained 9 ng of gp55-related protein per mg of cellular proteins and dexamethasonestimulated cells contained about five times MuMTV(RII1) cDNA and C57 lactating this amount. mammary gland RNA occurs is about 7” Figure 6 shows that considerable quan- lower than the T,,, for homologous hybrids tities of MuMTV were released into the [MuMTV(RIII) cDNA . MuMTV(RII1) culture medium as measured by molecular RNA]. Therefore, we presumed that, in hybridization assay (Ringold et al., 1975; order to determine whether MuMTVs proVaidya et al., 1976). Infected C57MG cells, duced by infected C57MG cells were the when not stimulated with dexamethasone, progeny of infecting virus or induced enreleased on an average 80 MuMTV virions dogenous C57 virus or both, thermal denaper cell in 24 h, whereas 240 virions per cell turation profiles would provide helpful inper 24 h were released after stimulation formation. However, as shown in Fig. 8, with dexamethasone. AT,,, for hybrids between C57 lactating Electron microscopic examination indi- mammary gland RNA and MuMTV(RII1) cated complete virion assembly and release cDNA was found to be only -3”. The T,,, by infected C57MG cells (Fig. 7). Morphovalues for hybrids between MuMTV(RII1) logically, virions released by C57MG cells cDNA and RNA from RI11 lactating mamwere indistinguishable from standard mary glands, C57 lactating mammary MuMTV; the characteristic surface glands and infected C57MG cells were 70”, “spikes” were clearly visible. 67”, and 68.5”, respectively. These values are very close to each other; therefore, we Thermal Denaturation Experiments conclude that thermal denaturation profiles Varmus et al. (1973) have reported that are inadequate to determine the origin of the temperature at which 50% denatur- the MuMTV produced by infected C57MG ation (T,) of the hybrids between cells.
MuMTV
INFECTION
MOUSE
FIG. 8. Thermal denaturation of hybrids. RNAs purified from infected C57MG cells (+), C57BL/6 lactating mammary glands (O), and RI11 lactating mammary glands (0) were hybridized with about 7000 cpm of MuMTV(RII1) cDNA. The nucleic acids were ethanol-precipitated and resuspended in 0.02 M Tris’ HCl, pH 7.4. Aliquots were incubated at the indicated temperature for 10 min and the extent of denaturation was measured by digestion with Z-31nuclease.
Titration
of MuMTV
in C57MG Cells
Our previous experience has shown that a successful MuMTV infection of nonmu-
19
CELLS
7. An electron micrograph of infected C57MG cells showing virions with the cell membrane. Large, prominent “spikes” are visible on viral envelope
FIG.
from
OF
MuMTV (arrows).
morphology
budding
rine cells requires an extremely high multiplicity of infection (m.0.i.) (Vaidya et al., 1976), and this has been confirmed by others (Ringold et al., 1977a; Howard et al., 1977). The inefficient infection of heterologous cells could have been due to a restriction of penetration by MuMTV in these cells which may lack appropriate receptors for the virus. However, as Fig. 9 shows, MuMTV infection of C57MG cells also seems to require a high m.o.i. In this experiment, C57MG cells were infected with m.o.i.‘s ranging from 4 X 10’ to 4 X lo” virions per cell and the level of MuMTV infection was determined by measuring the average number of MuMTV specific RNA molecules present per cell 5 weeks after the infection. The interval of 5 weeks insured that few, if any, of the inoculum virions would remain associated with the cells. Although it is difficult to rule out the possibility that a cell-to-cell spread of the virus would have occurred, Fig. 9 clearly shows that even at the highest m.o.i., a plateau level of infection was not achieved. However, even at m.0.i. of 400 virions per cell, MuMTV-specific RNA was detectable, al-
20
VAIDYA
FIG. 9. Titration of MuMTV on C57MG cells. About 10” C57MG cells each were infected with varying m.o.i. of MuMTV (range, 4 x lo’-4 x lo” virions per cell) and allowed to grow for 5 weeks. The cells were fed every 48 hr and passaged twice during the period. Dexamethasone at lo-” M was added 48 hr before the harvesting of cells. RNAs were extracted from the cells and the average number of MuMTV RNA molecules per cell was determined by hybridization with MuMTV cDNA as described for Fig. 4.
beit at a very low level. Other workers have obtained similar results when using heterologous cells (Ringold et al., 1977; Howard et al., 1977). It is not clear whether this relationship between decreasing m.o.i. and diminishing MuMTV RNA synthesis by the infected cells is due to a lower level of infection of every cell or due to a decrease in the fraction of cells infected, or both. When these cells were tested for MuMTV antigen expression by immunofluorescence, we did observe a decline in the number of cells showing MuMTV-specific fluorescence as a function of decreasing m.o.i. (data not shown). However, the correlation was neither linear nor logarithmic. DISCUSSION
In this paper we have shown that an epithelioid cell line derived from the mammary glands of a C57BL/6 mouse is susceptible to infection by MuMTV. Although nonmurine cells have been shown previously to be able to support infection by MuMTV (Vaidya et al., 1976; Lasfargues et al., 1976; Ringold et al., 1977a; Howard et al., 1977), the fact that the putative in viva target cells for MuMTV can be infected with the virus in vitro provides an addi-
ET AL.
tional resource to investigate the biology of MuMTV. Following infection with MuMTV, C57MG cells acquire additional proviral DNA sequences (Fig. 2). However, unlike the nomnurine cells (Ringold et al., 1977b; Vaidya, unpublished observations), most, if not all, of the additional MuMTV DNA sequences seem to be integrated into the host genome (Fig. 3). This phenomenon appears to be analogous to ASV-infected homologous and heterologous cells in that, although no unintegrated viral DNA can be detected in chronically infected chicken cells, such molecules are present in ASVinfected heterologous quail and duck cells (Guntaka et al., 1976). It is possible that this may be a feature of ASV and MuMTV infection of heterologous hosts. The endogenous MuMTV genome of uninfected C57MG cells appears to be unexpressed, as no viral RNA or protein could be detected even after the treatment with dexamethasone. MuMTV-infected cells, on the other hand, did synthesize viral RNA and proteins and this was stimulated by incorporation of dexamethasone in the medium, once again demonstrating the regulatory effect of glucocorticoids on MuMTV gene expression. Considering that dexamethasone was unable to induce MuMTV synthesis in uninfected cells, it is possible that a constitutive level of MuMTV RNA synthesis is required for dexamethasonemediated stimulation. It is not clear whether the virions expressed by infected C57MG cells were the progeny of the infecting virus, of the endogenous C57BL/6 MuMTV, or they were a mixture of both. Because there are at present no good markers for these viruses, we attempted to resolve this question by measuring AT,,, for the hybrids between MuMTV(RII1) cDNA and infected C57MG RNA. However, since AT,,, for heterologous hybrids, i.e., MuMTV(RII1) cDNA and Ct7BL/6 lactating mammary gland RNA, was only -3’, the thermal denaturation profiles were not helpful in resolving the origin of MuMTV expressed by infected C57MG cells. It was observed previously that optimum levels of infection by MuMTV in nonmurine cells could be achieved only when using
MuMTV
INFECTION
extremely high multiplicities of infection (Vaidya et al., 1976; Lasfargues et al., 1976b; Ringold et al., 1977a; Howard et al., 1977). The low efficiency of infection could have been due to use of heterologous cells. However, we have described here that even in homologous cells, the MuMTV infection process is not very efficient. Zivada et al. (1977) have recently obtained pseudotypes of vesicular stomatitis virus (VSV) that contained the VSV genome with MuMTV envelope proteins. VSV(MuMTV) pseudotypes were able to establish efficient infection of susceptible cells. ZBvada et al. (1977) presented evidence showing that there were two types of VSV(MuMTV) pseudotypes obtained, one of which was able to grow in heterologous cells (mink lung) but not in homologous cells (normal mouse mammary glands). They suggested that there were xenotropic and ecotropic MuMTVs present in mouse cells and that, because MuMTV infection of heterologous cells was reported to be inefficient (Vaidya et al., 1976; Lasfargues et al., 1976), only the xenotropic MuMTV was involved in infection of heterologous cells. If the concentration of the putative xenotropic MuMTV was low in the virus preparations, this would explain the inefficiency of MuMTV infection of heterologous cells. However, as described in this paper, comparable efficiency, or the lack of it, observed for the infection of homologous mouse cells with MuMTV would suggest that a mechanism other than the xenotropism or the ecotropism of the virus is responsible for the low efficiency of infection. Also, since VSV(MuMTV) pseudotype virions, unlike MuMTV, are able to establish efficient infection of susceptible cells, the restriction for MuMTV infection of cultured cells may lie at a postpenetration stage. We would like to point out that MuMTV virus preparations that are currently used in all investigations are uncloned because they are derived from primary biological sources (milk or cultured mammary tumor cells); these virions are probably the progeny of “undiluted” passage of virus from mother to offspring via milk. The possibility that such uncloned MuMTV preparations may contain large numbers of defective virions should be kept in mind.
OF
MOUSE
CELLS
21
Finally, we have been unable to observe any morphological or growth chracteristic changes in C57MG cells infected with MuMTV. However, in vitro correlates established for malignant transformation of cultured fibroblastic cells need not always apply to epithelial cells, and such correlates will have to be established more firmly for epithelial cells before the effect of MuMTV infection on epithelial cell transformation could be ascertained. ACKNOWLEDGMENTS We thank Dr. Carole Long for her critical reading of the manuscript and Jennie Lasfargues and Robert McCaffrey for assistance. This work was supported by United States Public Health Service Grants CA-08740 and CA-08515 and Contract NOl-CP-5356 within the Virus Cancer Program of the National Cancer Institute. REFERENCES DROHAN, W., KETTMEN, R., COLCHER, D., and SCHLOM, J. (1977). Isolation of the mouse mammary tumor virus sequences not transmitted as geminal provirus in the C3H and RI11 mouse strains. J. Virol. 21,986X%5. GUNTAKA, R. R., RICHARDS, 0. C. SHANK, P. R., KUNG, H. J., DAVIDSON, N., FRITSCH, E., BISHOP, J. M., and VARMUS, H. E. (1976). Covalently closed circular DNA of avian sarcoma virus: Purification from nuclei of infected quail tumor cells and measurement by electron microscopy and gel electrophoresis. J. Mol. Biol. 106, 337-357. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369. HOWARD, D. K., COLCHER, D., TERAMOTO, Y. A., YOUNG, J. M., and SCHLOM, J. (1977). Characterii&ion of mouse mammary tumor viruses propagated in heterologous cells. Cancer Res. 37, 2696-2704. LASFARGUES, E. Y., KRAMARSKY, B., LASFARGUES, J. C., and MOORE, D. H. (1974). Detection of mouse mammary tumor virus in cat cells infected with purified B-particles from RI11 milk. J. Nat. Cancer Inst. 53, 1831-1833. LASFARGUES, E. Y., VAIDYA, A. B., LASFARGUES, J. C., and MOORE, D. H. (1976a). In vitro susceptibility of mink lung cells to the mouse mammary tumor virus. J. Nat. Cancer Inst. 57,447-449. LASFARGUES, E. Y., LASFARGUES, J. C., DION, A. S., GREENE, A. E. and MOORE, D. H. (1976b). Experimental infection of a cat kidney cell line with mouse mammary tumor virus. Cancer Res. 30, 167-178. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with Folin phenol reagent. J. Biol. Chem. 192, 265-275. MICHALIDES, R., and SCHLOM, J. (1975). Relationship
22 in nucleic acid sequences between tumor virus variants. Proc. Nat. 4635-4639. MORRIS, V. L., MEDEIROS, E., BISHOP, J. M., and VARMUS, H. ison of mouse mammary tumor in inbred, wild, and Asian mice, normal organs from inbred mice.
VAIDYA mouse mammary Acad. Sci. USA 72, RINGOLD, G. M., E. (1977). Compar virus-specific DNA and in tumors and J. Mol. Biol. 114,
73-92.
MUTO, M. (1977). Estimation of gene reiteration from hybridization kinetics in moderate deoxyribonucleic acid excess. Biochem. J. 165, 19-25. PARKS, W. P., RANSOM, J. C., YOUNG, H. A., and SCOLNICK, E. M. (1975). Mammary tumor virus induction by glucocorticoids: Characterization of specific transcriptional regulation. J. Biol. Chem. 250,3330-3336.
RINGOLD, G. M., LASFARGUES, E. Y., BISHOP, J. M., and VARMUS, H. E. (1975). Production of mouse mammary tumor virus by cultured cells in absence and presence of hormones: Assay by molecular hybridization. Virology 65, 135-147. RINGOLD, G. M., CARDIFF, R. D., VARMUS, H. E., and YAMAMOTO, K. R. (1977a). Infection of cultured rat hepatoma cells by mouse mammary tumor viurs. Cell 10, 11-18. RINGOLD, G. M., YAMAMOTO, K. R., SHANK, P. R., and VARMUS, H. E. (1977b). Mouse mammary tumor virus DNA in infected rat cells: Characterization of unintegrated forms. Cell 10, 19-26. SHANK, P. R., COHEN, J. C., VARMUS, H. E., YAMAMOTO, K. R., and RINGOLD, G. M. (1978). Mapping of linear and circular forms of mouse mammary tumor virus DNA with restriction endonucleases: Evidence for a large specific deletion occurring at high frequency during circularization. Proc. Nat. Acad. Sci. USA, 75,2112-2116. SHEFFIELD, J. B., DALY, T., DION, A. S., and TARASCHI, N. (1977). Procedures for radioimmunoassay of the mouse mammary tumor virus. Cancer Res. 37,
ET
AL
1480-1485. SLEMMER, G. L. (1974). Interactions of separate types of cells during normal and neoplastic mammary gland growth. J. Invest. Dermatol. 63, 27-47. TERAMOTO, Y. A., KUFE, D., and SCHLOM, J. (1977). Multiple antigenic determinants on the major surface glycoprotein of murine mammary tumor viurses. Proc. Nat. Acad. Sci. USA 74, 3564-3568. TIBBETTS, C., JOHANSSON, K., and PHILIPSON, L. (1973). Hydroxyapatite chromatography and formamide denaturation of adenovirus DNA. J. Viral. 12,218-225.
VAIDYA, A. B., BLACK, M. M., DION, A. S., and MOORE, D. H. (1974). Homology between human breast tumor RNA and mouse mammary tumor virus genome. Nature 249, 565-567. VAIDYA, A. B., LASFARGUES, E. Y., HEUBF,L, G., LASFARGUES, J. C., and MOORE, D. H. (1976). Murine mammary tumor virus: Characterization of infection of nonmurine cells. J. Virol. 18, 911-917. VAIDYA, A. B., and LASFARGUES, E. Y. (1977). Murine mammary tumor virus infection of mouse mammary epithelial cells in vitro. Proc. Amer. Assoc. Cancer Res. 18, 240. VARMUS, H. E., BISHOP, J. M., NOWINSKY, R. C., and SARKAR, N. H. (1972). Mammary tumor virus-specific nucleotide sequences in mouse DNA. Nature [New Biol.] 238, 189-191. VARMUS, H. E., QUINTRELL, N., NOWINSKY, R., MEDEIROS, E., SARKAR, N. H., and BISHOP, J. M. (1973). Characterization of mouse mammary tumor virusspecific RNA in murine tissues. J. Mol. Biol. 79, 663-679. Z~~VADA, J., DICKSON, C., and WEISS, R. (1977). Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus. Virology 82, 221-231. ZIMMERMAN, S. B., and SANDEEN, L. (1966). The ribonuclease activity of crystallized pancreatic deoxyribonuclease. Anal. Biochem. 14, 269-277.
90,
Murine
AKHIL
12-22
(1978)
Mammary Tumor Virus (MuMTV) Infection of an Epithelial Line Established from C57BL/6 Mouse Mammary Glands B. VAIDYA,’
ETIENNE Y. LASFARGUES, WILLIAM G. COUTINHO
Department of Microbiology and Immunology, The Hahnemann Pennsylvania 19102 and ins&&e for Medical Research, Accepted
June
JOEL
Medical Camden,
B. SHEFFIELD,’
Cell
AND
College, Philadelphia, h&w Jersey 08103
5, 1978
An epithelioid cell line derived from the mammary glands of a C57BL/6 mouse and designated C57MG cell line was found to be susceptible to infection by murine mammary tumor virus (MuMTV). Although uninfected C57MG cells contain endogenous MuMTVrelated DNA sequences, no RNA sequences homologous to MuMTV were detectable, even after treatment with the glucocorticoid, dexamethasone. However, after infection with MuMTV, these cells acquire additional MuMTV DNA, and viral RNA and proteins were readily detectable. Most, if not all, of the additional MuMTV DNA in infected C57MG cells appeared to be integrated. Synthesis of viral RNA and protein, and the release of virions into culture fluid by infected C57MG cells, was stimulated by incorporation of dexamethasone in the growth medium. The efficiency of infection by MuMTV in C57MG cells was similar to that in nonmurine cells. There was a direct relationship between multiplicity of infection (m.o.i.) and the average number of MuMTV RNA molecules detected per cell 5 weeks after infection. However, even at the highest m.o.i. used (4 X lo” virions/cell), a plateau in the average number of viral RNA molecules per cell was not achieved. The origin of MuMTV synthesized by infected C57MG cells, whether it is the progeny of infecting MuMTV or of the endogenous C57BL/6 MuMTV or of both, remains undetermined. We have been unable to detect anv mornholo~icai or growth pattern changes in C57MG cells following infection with MuM’k’. -
(Ringold et al., 1977a) and Howard et al. (1977) have confirmed the susceptibility of cat kidney and mink lung cells to infection by MuMTV. Several interesting features have emerged from MuMTV infection of nonmurine cells, which include the following. (1) The amount of MuMTV RNA present in nonmurine cells is also stimulated by glucocorticoids (Vaidya et al., 1976; Ringold et al., 1977a), just as it is in cultures derived from mouse mammary tumors that synthesize MuMTV (Ringold et al., 1975; Parks et al., 1975). This would indicate that either MuMTV proviral DNA integrates in nonmurine cells at a site adjacent to the sequences that are recognized by the glucocorticoid receptor complex or that MuMTV proviral DNA itself carries such recognition sequences. (2) When stimulated with glucocorticoids, infected nonmurine cells ac-
INTRODUCTION
A detailed analysis of the biology of murine mammary tumor virus (MuMTV), the only retravirus known whose main pathological manifestation is a hormonally controlled carcinoma, has been hampered for many years due to the lack of appropriate in vitro systems. Until recently, a major block had been the inability to infect cultured cells with MuMTV. We have reported previously that epithelioid cell lines derived from cat kidney and mink lung are susceptible to infection by MuMTV (Lasfargues et al., 1974, 1976a, b; Vaidya et al., 1976). This observation was later extended to a cell line derived from a rat hepatoma ’ To whom reprint requests should be addressed at the Department of Microbiology and Immunology, the Hahnemann Medical College, Philadelphia, Pa. 19102. ’ Present address: Department of Biology, Temple University, Philadelphia, Pa. 19122. 12 0042-6822/78/0901-0012$02.00/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
MuMTV
INFECTION
cumulate unintegrated MuMTV DNA molecules in linear as well as in covalently closed circular duplex forms (Ringold et al., 197713; Vaidya, unpublished observations). Such molecules have been used in generating a restriction endonuclease cleavage map of MuMTV (Shank et aZ., 1978). And, (3) infection of nomnurine cells has provided progeny virions for use in comparative studies of viral proteins obtained from different strains of MuMTV so as to rule out host-mediated modification (Teramoto et al., 1977). However, appropriate biological studies on MuMTV can best be carried out when its target cell, i.e., the mammary epithelium of mouse, is infected with the virus. In this paper, we describe MuMTV infection of an epithelioid cell line established from the mammary glands of a C57BL/6 mouse. A preliminary report of these results was presented at the 68th Annual Meeting of the American Association for Cancer Research in Denver, Colorado (Vaidya and Lasfargues, 1977). MATERIALS
AND
METHODS
Cells and virus. A cell line, designated C57MG, was established by one of us (W.G.C.) from the mammary glands of a 23-week-old C57BL/6 female mouse. The mammary glands were dissociated overnight with 200 units/ml of collagenase (Worthington) and epithelial cells were separated on a O-50% Ficoll (Pharmacia) gradient by centrifugation at 8000 g for 1 hr. The cells were epithelial in morphology and were grown in Hanks’-Eagle’s Minimal Essential Medium (HE-MEM) containing 10 pg/ml insulin and 10% fetal bovine serum. MuMTV was purified from the milk of the RI11 strain of mice by sucrose density gradient. For the preparation of inoculum, purified virions were resuspended in HEMEM and passed through a 0.45~pm membrane filter that was pretreated with a 1% solution of polyvinylpyrrolidone-K-90 (GAF Corp.). The concentration of virions was determined by molecular hybridization assay as described below. Infection with MuMTV. Infection of C57MG cells was carried out using the techniques described previously for nonmurine
OF
MOUSE
CELLS
13
cells (Lasfargues et al., 1976; Vaidya et al., 1976). Briefly, about lo6 cells were resuspended per ml of HE-MEM containing 4 pg/ml polybrene (Aldrich) and about 10” MuMTV particles per ml. After 1 hr at 37” with intermittent agitation, the cell-virus suspension was diluted with complete medium and plated on culture flasks. For the experiments involving titration of MuMTV, constant numbers of cells were infected with logarithmic dilutions of MuMTV. Extraction of nucleic acids. Cultured cells were lysed in extraction buffer (0.05 M Tris-HCl, pH 8.0, 0.02 M EDTA, 0.1 M NaCl, and 0.5% sodium dodecyl sulfate) at a concentration of about lo7 cells/ml and treated with 200 pg/ml of proteinase K (EM Laboratories) at 37” for 90 min. After two extractions with buffer-saturated phenol, nucleic acids were precipitated with ethanol and stored at -20”. To purify DNA, nucleic acids were resuspended in 3 mM EDTA, pH 7.0, and adjusted to 0.3 M NaOH. Following incubation in a boiling water bath for 20 min and neutralization with HCl, DNA was precipitated with cold ethanol. The alkali treatment hydrolyzed RNA and reduced the size of DNA chains to 300 to 400 nucleotides long (Tibbetts et al., 1973). To purify RNA, nucleic acids were resuspended in DNase buffer (0.02 M Tris-HCl, pH 7.4 and 0.01 M MgC12) and treated with 20 yg/ml of iodoacetate-treated (Zimmerman and Sandeen, 1966) RNase-free deoxyribonuclease I (Worthington) for 2 hr at room temperature. The solution was adjusted to 0.05 M Tris-HCl, pH 8.0, and 0.01 M EDTA, and then extracted twice with buffer-saturated phenol and precipitated with ethanol. Both DNA and RNA were resuspended at a concentration of >5 mg/ml in 3 mJ4 EDTA or 0.02 M Tris-HCl, pH 7.4, respectively. Separation of high molecular weight cellular DNA from low molecular weight unintegrated DNA was carried out according to the procedure of Hirt (1967). The cells were grown in roller bottles and lysed in situ with a solution containing 0.02 M TrisHCl, pH 7.5, 0.01 M EDTA and 0.6% SDS at a concentration of 0.5-l X lo7 cells/ml. After incubation on a roller apparatus for 5-10 min, the lysate was gently scraped off
14
VAIDYA
and made 1 M in NaCl by slow addition of 5 M NaCl with gentle mixing. The lysate was stored at 4’ overnight and the coagulate of high molecular weight DNA, protein and SDS was pelleted out by centrifugation at 15,000 g for 30 min. The pellet and the supernatants were separated and DNA was extracted as described above. RNA was extracted from lactating mammary glands of mice by homogenization of the glands in a Virtis homogenizer, followed by phenol extraction as described earlier (Vaidya et al., 1974). MuMTV cDNA. Highly radioactive DNA complementary to MuMTV RNA was synthesized by detergent-disrupted purified virions utilizing the endogenous RNA-directed DNA polymerase in the presence of actinomycin D as described before (Vaidya et al., 1974), and purified by batch elution from hydroxylapatite (BioRad). The specific activity of the cDNA was about 2 X lo7 cpm/pg. All the different batches of cDNA synthesized were specific and gave consistent results as judged by cDNA MuMTV 70 S RNA hybridization kinetics. Although a large proportion of the cDNA may be a multiple transcript of a small segment of the viral genome, we have found that when hybridization was conducted at a cDNA to MuMTV RNA ratio of 4, about 40% of the RNA was protected from RNase digestion under high salt conditions. The cDNAs synthesized by us have served as sensitive and accurate probes for the detection and quantitation of MuMTV nucleic acids. Molecular hybridization. Cellular DNA to MuMTV cDNA annealings were performed in 50 ~1 volume in conical polypropylene tubes. Various amounts of cellular DNA were mixed with about 1000 cpm of [3H]cDNA and adjusted to 0.6 M NaCl, 0.04 M Tris HCl, pH 7.2, and 0.002 M EDTA by addition of a concentrated buffer salt solution. The annealing mixtures were adjusted to a constant DNA content by addition of sheared salmon sperm DNA and covered with mineral oil. After incubation at 100’ for 10 min and quick chilling on ice, annealing was conducted at 68” for 66 hr. The extent of annealing was determined by resistance to single strand-specific nuclease
ET AL.
S1 as described previously (Vaidya et al., 1976). When using the low molecular weight DNA associated with Hirt supernatants, volumes that were equivalent to the volumes used for Hirt pellet DNA were employed in the annealing reactions. Hybridization of RNA to MuMTV cDNA was carried out in a lo-y1 volume as described (Vaidya et al., 1974, 1976). The average number of MuMTV RNA molecules per cell was estimated by comparing Crtljs (C,t at which half-maximum hybridization occurs) for the sample RNA with C,.tl,z for MuMTV 70 S RNA. Quantitation of MuMTV by molecular hybridization assay. The number of MuMTV particles in a sample was measured by molecular hybridization assay as described (Ringold et al., 1975; Vaidya et al., 1976). After pelleting the virions by centrifugation, RNA was purified by proteinase-K treatment and phenol extractions followed by ethanol precipitation in the presence of yeast RNA as a carrier and hybridized with MuMTV cDNA. The kinetics of hybridization was plotted as a function of VOt (the product of volume of sample in milliliters and time of incubation in hours). The concentration of MuMTV RNA was determined by obtaining Votl,z value ( Vat at which half-maximum hybridization occurred) and the virion content of the sample was estimated on the assumption that each mature MuMTV particle contains one 70 S RNA molecule. Thermal denaturation of hybrids. Approximately 10,000 cpm of MuMTV cDNA were hybridized to saturation with RNA from various sources. The hybrids were ethanol-precipitated and resuspended in 0.02 M Tris. HCl, pH 7.4. Aliquots of the hybrids were incubated at various temperatures for 10 min and the extent of denaturation was measured by digestion with S1 nuclease. Radioimmunoassay (RIA). The procedure for RIA detection and quantitation of gp55, the major glycoprotein of MuMTV, has been described elsewhere (Sheffield et al., 1977). Briefly, a cytosol preparation was made by disrupting the cells in a hypotonic buffer containing nonionic detergent NP-40 followed by a low-speed centrifugation to
MuM’I’V
15
1Nl”r;C;‘l’lUN
remove nuclei. The protein concentration of cytosols was determined by the method of Lowry et al. (1951) and the ability of this preparation to inhibit immunoprecipitation of ’ 51-labeled gp55 under limiting antibody concentration was measured. The concentration of gp55-related antigen was estimated by comparison with the immunoprecipitation inhibition curve generated by known quantities of unlabeled gp55. Electron microscopy. Cells growing in monolayers were fixed with 2.5% glutaraldehyde (pH 6.8) for 2 hr and rinsed with H20 overnight. After dehydration with graded ethanol, the monolayer of cells was removed by ethylene dichloride, washed with ethanol, and embedded in Epon-Ardldite mixture. Ultra-thin sections were cut with glass knives, mounted on copper grids, and stained with uranyl acetate and lead citrate. The sections were examined and photographed in a JEM-1OOB electron microscope.
RESULTS
C57MG
Cell Line
Mammary glands of mice contain three different types of epithelial cells, viz., alveolar, ductal, and myoepithelial. As shown by Slemmer (1974), these cell types have distinct heritable behavior and, in normal mammary glands, they do not seem to differentiate from a single progenitor stem cell The C57MG cell line was derived from the mammary glands of a 23-week-old retired C57BL/6 breeder. As shown in Fig. 1, the cells were of epithelial morphology, the origin of which was not determined. However, it is quite likely that the epithelial cells of C57MG cell line are either cells of alveolar, ductal, or myoepithelial origin, or a mixture of two or more types. The cells used for experiments described in this paper were uncloned; therefore, we cannot rule out the possibility that we were dealing with a mixed population of epithelial cells.
FIG. 1. Photomicrographs of C57MG cells growing in a monolayer. A, the cells at confluency showing epithelial morphology. B, a higher magnification micrograph of cells that have not reached confluency. Note the spreading on the substratum, which is typical of epithelial cells.
16
VAIDYA
After infection with MuMTV, we did not observe any morphological or growth characteristic changes in C57MG cells. In fact, uninfected and chronically infected C57MG cells were distinguishable only by means of MuMTV production by the infected cells as described below. Increase in MuMTV Prouiral Following Infection
Sequences
It has been recognized for some time that all cells of Mus musculus contain multiple of DNA sequences related to copies MuMTV (Varmus et al., 1972; Michelides and Schlom, 1975), although there may be some quantitative (Morris et al., 1977) or qualitative (Drohan et al., 1977) differences among different strains or cells from normal organs and mammary tumors. As shown in Fig. 2, C57MG cells also contain DNA sequences related to MuMTV as determined by annealing experiments using MuMTV cDNA. After infection with MuMTV, C57MG cells acquired additional MuMTV DNA sequences (Fig. 2). In this experiment, increasing amounts of DNA from C57MG control or infected cells were annealed to MuMTV cDNA in 50 ~1 volume for 66 hr. The total DNA content of the annealing reaction was kept constant (as a viscosity
Ld -
l l /
FIG. 2. Acquisition of additional MuMTV proviral DNA by infected C57MG cells. C57MG cells were infected with MuMTV at a high multiplicity and DNA was extracted from uninfected and chronically infected C57MG cells. Various amounts of cellular DNA were annealed with MuMTV cDNA, keeping the reaction volume and the time of incubation constant. The extent of annealing was determined by resistance of cDNA to S1 nuclease. (*). DNA from uninfected cells and (O), DNA from chronically infected cells.
ET
AL.
control) by adjustment with salmon sperm DNA. Fig. 2 shows that about 64% of MuMTV cDNA annealed to infected C57MG cellular DNA at the plateau of the curve as compared to about 45% annealed to uninfected C57MG DNA. In these experiments the driver DNA molecules, at the highest concentration, were only in moderate excess, and the differences in plateau levels observed for cDNA annealing to infected and uninfected C57MG DNA were perhaps due to the difference in driver DNA to cDNA ratios (Muto, 1977). About 10 pg of DNA from infected cells was sufficient to achieve half-maximum annealing with MuMTV cDNA under these conditions. In contrast, half-maximum annealing with C57MG DNA from uninfected cells required about 30 pg of DNA. Therefore, we estimate that the infection with MuMTV resulted in about a three-fold increase in MuMTV-related DNA sequences in the C57MG cells. In order to determine whether the additional MuMTV proviral DNA was integrated into the cellular DNA, we carried out extraction of cellular DNA according to the procedure of Hirt (1967). This procedure separates high molecular weight cellular DNA from unintegrated, relatively low molecular weight DNA. The cells were grown in the presence of lo-” M dexamethasone for 48 l-n-s before extraction. The hybridization procedure was similar to the one described for Fig. 2. After the Hirt extraction and purification, increasing amounts of high molecular weight DNA (Hirt pellet DNA) were annealed to MuMTV cDNA in a constant volume and identical conditions were used for the equivalent amounts (as per volume) of low molecular weight DNA (Hirt supernatant DNA). Figure 3 shows that Hirt pellet DNA from both uninfected and infected C57MG cells annealed to MuMTV cDNA to the extent shown in Fig. 2; that is, there was about a 3-fold increase in high molecular weight-associated MuMTV DNA after infection of C57MG cells with MuMTV. Hirt supernatant DNA showed a limited extent of annealing with MuMTV cDNA, but this was probably due to a low-level contamination of Hirt supernatant DNA with high molecular weight
MuMTV
1
10 iG HlRT PELLET
INFECTION
100
DNA OR ITS EWYRLENT
HlR,
SUPERHATIWT
FIG. 3. Most, if not all, of the additional MuMTV proviral DNA in infected C57MG cells is associated with high molecular weight cellular DNA. The procedure of Hirt (1967) was used to separate high molecular weight cellular DNA (Hirt pellet DNA) from low molecular weight unintegrated DNA (Hirt supernatant DNA) from both uninfected and chronically infected C57MG cells grown in the presence of IO-” it4 dexamethasone. Annealing with MuMTV cDNA was performed using various amounts of Hirt pellet DNA or its equivalent volume of Hirt supernatant DNA as described in Materials and Methods. Symbols: 0, 0, Hirt pellet and Hirt supernatant DNA, respectively, from chronically infected C57MG cells; +, 0 Hirt pellet and Hirt supernatant DNA, respectively, from uninfected C57MG cells.
DNA. Although these results are indicative of integrated MuMTV DNA in infected C57MG cells, covalent linkage of viral DNA with the host genome is not rigorously demonstrated. However, it does seem that most, if not all, of the increased MuMTV DNA sequences in infected C57MG cells are associated with high molecular weight DNA of the host. In similar experiments, it was determined that in MuMTV-infected nonmurine cells like rat hepatoma cells (Ringold et al., 1977b), cat kidney and mink lung cells (Vaidya, unpublished observations), some of the proviral sequences were present in unintegrated forms, in addition to the integrated MuMTV provirus. This is comparable to the avian system in which chicken cells infected with avian sarcoma virus (ASV) do not carry unintegrated provirus, but ASV-infected duck and quail cells do (Guntaka et al., 1976). MuMTV Synthesis Cells
by Infected
C57MG
No detectable MuMTV RNA synthesis is observed in uninfected C57MG cells even after treatment with dexamethasone. This
OF
MOUSE
CELLS
17
is in contrast to the finding of Varmus et al. (1973) and our own observation that lactating mammary glands of C57BL mice contain about 40 copies of MuMTV RNA per cell, even though no viral antigens or virions are produced. The reason for the lack of MuMTV RNA in C57MG cells may be that, since the cell line was established from the mammary glands of a nonlactating mouse, no transcription of the MuMTV genes may occur under these physiologic conditions. In contrast, C57MG cells infected with MuMTV synthesized MuMTV RNA; when not stimulated with dexamethasone, about 150 copies of MuMTV RNA were present per cell, on an average (Fig. 4). Inclusion of dexamethasone in the culture medium enhanced the concentration of MuMTV RNA lo-fold (Fig. 4). The fact that these MuMTV specific RNA molecules were engaged in viral protein synthesis was demonstrated by measuring the amount of the major viral glycoprotein, gp55, in these cells by radioimmunoassay. Figure 5 shows that, whereas uninfected C57MG cells were not synthesizing any detectable gp55-related antigens whether treated with dexamethasone or
FIG. 4. Intracellular concentration of MuMTV RNA in infected C57MG cells in the presence or absence of dexamethasone. About 10” C57MG cells infected with MuMTV were treated with 1O-5 M dexamethasone for 48 hr and total cellular RNA was purified from both treated and untreated infected C57MG cells. Kinetics of cellular RNA to MuMTV cDNA hybridization were performed as described in Materials and Methods. Intracellular concentration of MuMTV RNA in infected C57MG cells increased about lo-fold in cells treated with dexamethasone.
18
VAIDYA
ut
FIG. 5. Radioimmunoassay
COMPETING
ET
AL.
PROTEIN
(RIA) for the major glycoprotein of MuMTV, gp55, in infected and uninfected C57MG cells. The cytoplasmic protein was obtained from infected and uninfected C57MG cells that were grown in the absence (open symbols) or presence (closed symbols) of 10 ’ M dexamethasone for 48 hr. The ability of various amounts of the protein to inhibit precipitation of ““I-labeled gp55 by limiting concentration of anti-gp55 was measured as described in Materials and Methods. Symbols: +, 0, protein from infected C57MG cells; 0, 0 protein from uninfected C57MG cells.
FIG. 6. Release of MuMTV by infected C57MG cells grown in the presence of the absence of dexamethasone. Confluent cells were either left untreated or exposed to lo-” M dexamethasone for 48 hr after which the virions present in a known volume of the culture fluids were recovered by centrifugation. The RNA was purified and resuspended in a small volume of 0.02 M Tris. HCl, pH 7.4. Various dilutions of this RNA were hybridized in a lo-p1 volume with MuMTV cDNA for 20 hr and the extent of hybridization assayed by S1 nuclease. The percentage hybridization was plotted as a function Vd as described in Materials and Methods. Symbols: 0, cells grown in the absence of dexamethasone; +, growth in the presence of dexamethasone.
not, the infected cells when unstimulated contained 9 ng of gp55-related protein per mg of cellular proteins and dexamethasonestimulated cells contained about five times MuMTV(RII1) cDNA and C57 lactating this amount. mammary gland RNA occurs is about 7” Figure 6 shows that considerable quan- lower than the T,,, for homologous hybrids tities of MuMTV were released into the [MuMTV(RIII) cDNA . MuMTV(RII1) culture medium as measured by molecular RNA]. Therefore, we presumed that, in hybridization assay (Ringold et al., 1975; order to determine whether MuMTVs proVaidya et al., 1976). Infected C57MG cells, duced by infected C57MG cells were the when not stimulated with dexamethasone, progeny of infecting virus or induced enreleased on an average 80 MuMTV virions dogenous C57 virus or both, thermal denaper cell in 24 h, whereas 240 virions per cell turation profiles would provide helpful inper 24 h were released after stimulation formation. However, as shown in Fig. 8, with dexamethasone. AT,,, for hybrids between C57 lactating Electron microscopic examination indi- mammary gland RNA and MuMTV(RII1) cated complete virion assembly and release cDNA was found to be only -3”. The T,,, by infected C57MG cells (Fig. 7). Morphovalues for hybrids between MuMTV(RII1) logically, virions released by C57MG cells cDNA and RNA from RI11 lactating mamwere indistinguishable from standard mary glands, C57 lactating mammary MuMTV; the characteristic surface glands and infected C57MG cells were 70”, “spikes” were clearly visible. 67”, and 68.5”, respectively. These values are very close to each other; therefore, we Thermal Denaturation Experiments conclude that thermal denaturation profiles Varmus et al. (1973) have reported that are inadequate to determine the origin of the temperature at which 50% denatur- the MuMTV produced by infected C57MG ation (T,) of the hybrids between cells.
MuMTV
INFECTION
MOUSE
FIG. 8. Thermal denaturation of hybrids. RNAs purified from infected C57MG cells (+), C57BL/6 lactating mammary glands (O), and RI11 lactating mammary glands (0) were hybridized with about 7000 cpm of MuMTV(RII1) cDNA. The nucleic acids were ethanol-precipitated and resuspended in 0.02 M Tris’ HCl, pH 7.4. Aliquots were incubated at the indicated temperature for 10 min and the extent of denaturation was measured by digestion with Z-31nuclease.
Titration
of MuMTV
in C57MG Cells
Our previous experience has shown that a successful MuMTV infection of nonmu-
19
CELLS
7. An electron micrograph of infected C57MG cells showing virions with the cell membrane. Large, prominent “spikes” are visible on viral envelope
FIG.
from
OF
MuMTV (arrows).
morphology
budding
rine cells requires an extremely high multiplicity of infection (m.0.i.) (Vaidya et al., 1976), and this has been confirmed by others (Ringold et al., 1977a; Howard et al., 1977). The inefficient infection of heterologous cells could have been due to a restriction of penetration by MuMTV in these cells which may lack appropriate receptors for the virus. However, as Fig. 9 shows, MuMTV infection of C57MG cells also seems to require a high m.o.i. In this experiment, C57MG cells were infected with m.o.i.‘s ranging from 4 X 10’ to 4 X lo” virions per cell and the level of MuMTV infection was determined by measuring the average number of MuMTV specific RNA molecules present per cell 5 weeks after the infection. The interval of 5 weeks insured that few, if any, of the inoculum virions would remain associated with the cells. Although it is difficult to rule out the possibility that a cell-to-cell spread of the virus would have occurred, Fig. 9 clearly shows that even at the highest m.o.i., a plateau level of infection was not achieved. However, even at m.0.i. of 400 virions per cell, MuMTV-specific RNA was detectable, al-
20
VAIDYA
FIG. 9. Titration of MuMTV on C57MG cells. About 10” C57MG cells each were infected with varying m.o.i. of MuMTV (range, 4 x lo’-4 x lo” virions per cell) and allowed to grow for 5 weeks. The cells were fed every 48 hr and passaged twice during the period. Dexamethasone at lo-” M was added 48 hr before the harvesting of cells. RNAs were extracted from the cells and the average number of MuMTV RNA molecules per cell was determined by hybridization with MuMTV cDNA as described for Fig. 4.
beit at a very low level. Other workers have obtained similar results when using heterologous cells (Ringold et al., 1977; Howard et al., 1977). It is not clear whether this relationship between decreasing m.o.i. and diminishing MuMTV RNA synthesis by the infected cells is due to a lower level of infection of every cell or due to a decrease in the fraction of cells infected, or both. When these cells were tested for MuMTV antigen expression by immunofluorescence, we did observe a decline in the number of cells showing MuMTV-specific fluorescence as a function of decreasing m.o.i. (data not shown). However, the correlation was neither linear nor logarithmic. DISCUSSION
In this paper we have shown that an epithelioid cell line derived from the mammary glands of a C57BL/6 mouse is susceptible to infection by MuMTV. Although nonmurine cells have been shown previously to be able to support infection by MuMTV (Vaidya et al., 1976; Lasfargues et al., 1976; Ringold et al., 1977a; Howard et al., 1977), the fact that the putative in viva target cells for MuMTV can be infected with the virus in vitro provides an addi-
ET AL.
tional resource to investigate the biology of MuMTV. Following infection with MuMTV, C57MG cells acquire additional proviral DNA sequences (Fig. 2). However, unlike the nomnurine cells (Ringold et al., 1977b; Vaidya, unpublished observations), most, if not all, of the additional MuMTV DNA sequences seem to be integrated into the host genome (Fig. 3). This phenomenon appears to be analogous to ASV-infected homologous and heterologous cells in that, although no unintegrated viral DNA can be detected in chronically infected chicken cells, such molecules are present in ASVinfected heterologous quail and duck cells (Guntaka et al., 1976). It is possible that this may be a feature of ASV and MuMTV infection of heterologous hosts. The endogenous MuMTV genome of uninfected C57MG cells appears to be unexpressed, as no viral RNA or protein could be detected even after the treatment with dexamethasone. MuMTV-infected cells, on the other hand, did synthesize viral RNA and proteins and this was stimulated by incorporation of dexamethasone in the medium, once again demonstrating the regulatory effect of glucocorticoids on MuMTV gene expression. Considering that dexamethasone was unable to induce MuMTV synthesis in uninfected cells, it is possible that a constitutive level of MuMTV RNA synthesis is required for dexamethasonemediated stimulation. It is not clear whether the virions expressed by infected C57MG cells were the progeny of the infecting virus, of the endogenous C57BL/6 MuMTV, or they were a mixture of both. Because there are at present no good markers for these viruses, we attempted to resolve this question by measuring AT,,, for the hybrids between MuMTV(RII1) cDNA and infected C57MG RNA. However, since AT,,, for heterologous hybrids, i.e., MuMTV(RII1) cDNA and Ct7BL/6 lactating mammary gland RNA, was only -3’, the thermal denaturation profiles were not helpful in resolving the origin of MuMTV expressed by infected C57MG cells. It was observed previously that optimum levels of infection by MuMTV in nonmurine cells could be achieved only when using
MuMTV
INFECTION
extremely high multiplicities of infection (Vaidya et al., 1976; Lasfargues et al., 1976b; Ringold et al., 1977a; Howard et al., 1977). The low efficiency of infection could have been due to use of heterologous cells. However, we have described here that even in homologous cells, the MuMTV infection process is not very efficient. Zivada et al. (1977) have recently obtained pseudotypes of vesicular stomatitis virus (VSV) that contained the VSV genome with MuMTV envelope proteins. VSV(MuMTV) pseudotypes were able to establish efficient infection of susceptible cells. ZBvada et al. (1977) presented evidence showing that there were two types of VSV(MuMTV) pseudotypes obtained, one of which was able to grow in heterologous cells (mink lung) but not in homologous cells (normal mouse mammary glands). They suggested that there were xenotropic and ecotropic MuMTVs present in mouse cells and that, because MuMTV infection of heterologous cells was reported to be inefficient (Vaidya et al., 1976; Lasfargues et al., 1976), only the xenotropic MuMTV was involved in infection of heterologous cells. If the concentration of the putative xenotropic MuMTV was low in the virus preparations, this would explain the inefficiency of MuMTV infection of heterologous cells. However, as described in this paper, comparable efficiency, or the lack of it, observed for the infection of homologous mouse cells with MuMTV would suggest that a mechanism other than the xenotropism or the ecotropism of the virus is responsible for the low efficiency of infection. Also, since VSV(MuMTV) pseudotype virions, unlike MuMTV, are able to establish efficient infection of susceptible cells, the restriction for MuMTV infection of cultured cells may lie at a postpenetration stage. We would like to point out that MuMTV virus preparations that are currently used in all investigations are uncloned because they are derived from primary biological sources (milk or cultured mammary tumor cells); these virions are probably the progeny of “undiluted” passage of virus from mother to offspring via milk. The possibility that such uncloned MuMTV preparations may contain large numbers of defective virions should be kept in mind.
OF
MOUSE
CELLS
21
Finally, we have been unable to observe any morphological or growth chracteristic changes in C57MG cells infected with MuMTV. However, in vitro correlates established for malignant transformation of cultured fibroblastic cells need not always apply to epithelial cells, and such correlates will have to be established more firmly for epithelial cells before the effect of MuMTV infection on epithelial cell transformation could be ascertained. ACKNOWLEDGMENTS We thank Dr. Carole Long for her critical reading of the manuscript and Jennie Lasfargues and Robert McCaffrey for assistance. This work was supported by United States Public Health Service Grants CA-08740 and CA-08515 and Contract NOl-CP-5356 within the Virus Cancer Program of the National Cancer Institute. REFERENCES DROHAN, W., KETTMEN, R., COLCHER, D., and SCHLOM, J. (1977). Isolation of the mouse mammary tumor virus sequences not transmitted as geminal provirus in the C3H and RI11 mouse strains. J. Virol. 21,986X%5. GUNTAKA, R. R., RICHARDS, 0. C. SHANK, P. R., KUNG, H. J., DAVIDSON, N., FRITSCH, E., BISHOP, J. M., and VARMUS, H. E. (1976). Covalently closed circular DNA of avian sarcoma virus: Purification from nuclei of infected quail tumor cells and measurement by electron microscopy and gel electrophoresis. J. Mol. Biol. 106, 337-357. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369. HOWARD, D. K., COLCHER, D., TERAMOTO, Y. A., YOUNG, J. M., and SCHLOM, J. (1977). Characterii&ion of mouse mammary tumor viruses propagated in heterologous cells. Cancer Res. 37, 2696-2704. LASFARGUES, E. Y., KRAMARSKY, B., LASFARGUES, J. C., and MOORE, D. H. (1974). Detection of mouse mammary tumor virus in cat cells infected with purified B-particles from RI11 milk. J. Nat. Cancer Inst. 53, 1831-1833. LASFARGUES, E. Y., VAIDYA, A. B., LASFARGUES, J. C., and MOORE, D. H. (1976a). In vitro susceptibility of mink lung cells to the mouse mammary tumor virus. J. Nat. Cancer Inst. 57,447-449. LASFARGUES, E. Y., LASFARGUES, J. C., DION, A. S., GREENE, A. E. and MOORE, D. H. (1976b). Experimental infection of a cat kidney cell line with mouse mammary tumor virus. Cancer Res. 30, 167-178. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with Folin phenol reagent. J. Biol. Chem. 192, 265-275. MICHALIDES, R., and SCHLOM, J. (1975). Relationship
22 in nucleic acid sequences between tumor virus variants. Proc. Nat. 4635-4639. MORRIS, V. L., MEDEIROS, E., BISHOP, J. M., and VARMUS, H. ison of mouse mammary tumor in inbred, wild, and Asian mice, normal organs from inbred mice.
VAIDYA mouse mammary Acad. Sci. USA 72, RINGOLD, G. M., E. (1977). Compar virus-specific DNA and in tumors and J. Mol. Biol. 114,
73-92.
MUTO, M. (1977). Estimation of gene reiteration from hybridization kinetics in moderate deoxyribonucleic acid excess. Biochem. J. 165, 19-25. PARKS, W. P., RANSOM, J. C., YOUNG, H. A., and SCOLNICK, E. M. (1975). Mammary tumor virus induction by glucocorticoids: Characterization of specific transcriptional regulation. J. Biol. Chem. 250,3330-3336.
RINGOLD, G. M., LASFARGUES, E. Y., BISHOP, J. M., and VARMUS, H. E. (1975). Production of mouse mammary tumor virus by cultured cells in absence and presence of hormones: Assay by molecular hybridization. Virology 65, 135-147. RINGOLD, G. M., CARDIFF, R. D., VARMUS, H. E., and YAMAMOTO, K. R. (1977a). Infection of cultured rat hepatoma cells by mouse mammary tumor viurs. Cell 10, 11-18. RINGOLD, G. M., YAMAMOTO, K. R., SHANK, P. R., and VARMUS, H. E. (1977b). Mouse mammary tumor virus DNA in infected rat cells: Characterization of unintegrated forms. Cell 10, 19-26. SHANK, P. R., COHEN, J. C., VARMUS, H. E., YAMAMOTO, K. R., and RINGOLD, G. M. (1978). Mapping of linear and circular forms of mouse mammary tumor virus DNA with restriction endonucleases: Evidence for a large specific deletion occurring at high frequency during circularization. Proc. Nat. Acad. Sci. USA, 75,2112-2116. SHEFFIELD, J. B., DALY, T., DION, A. S., and TARASCHI, N. (1977). Procedures for radioimmunoassay of the mouse mammary tumor virus. Cancer Res. 37,
ET
AL
1480-1485. SLEMMER, G. L. (1974). Interactions of separate types of cells during normal and neoplastic mammary gland growth. J. Invest. Dermatol. 63, 27-47. TERAMOTO, Y. A., KUFE, D., and SCHLOM, J. (1977). Multiple antigenic determinants on the major surface glycoprotein of murine mammary tumor viurses. Proc. Nat. Acad. Sci. USA 74, 3564-3568. TIBBETTS, C., JOHANSSON, K., and PHILIPSON, L. (1973). Hydroxyapatite chromatography and formamide denaturation of adenovirus DNA. J. Viral. 12,218-225.
VAIDYA, A. B., BLACK, M. M., DION, A. S., and MOORE, D. H. (1974). Homology between human breast tumor RNA and mouse mammary tumor virus genome. Nature 249, 565-567. VAIDYA, A. B., LASFARGUES, E. Y., HEUBF,L, G., LASFARGUES, J. C., and MOORE, D. H. (1976). Murine mammary tumor virus: Characterization of infection of nonmurine cells. J. Virol. 18, 911-917. VAIDYA, A. B., and LASFARGUES, E. Y. (1977). Murine mammary tumor virus infection of mouse mammary epithelial cells in vitro. Proc. Amer. Assoc. Cancer Res. 18, 240. VARMUS, H. E., BISHOP, J. M., NOWINSKY, R. C., and SARKAR, N. H. (1972). Mammary tumor virus-specific nucleotide sequences in mouse DNA. Nature [New Biol.] 238, 189-191. VARMUS, H. E., QUINTRELL, N., NOWINSKY, R., MEDEIROS, E., SARKAR, N. H., and BISHOP, J. M. (1973). Characterization of mouse mammary tumor virusspecific RNA in murine tissues. J. Mol. Biol. 79, 663-679. Z~~VADA, J., DICKSON, C., and WEISS, R. (1977). Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus. Virology 82, 221-231. ZIMMERMAN, S. B., and SANDEEN, L. (1966). The ribonuclease activity of crystallized pancreatic deoxyribonuclease. Anal. Biochem. 14, 269-277.
