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Specific restriction of avian sarcoma viruses by a line of transformed lymphoid cells
89, X0-371
VIROLOGY
Specific
(1978)
Restriction
PAUL
of Avian
E. NEIMAN,
Sarcoma Lymphoid
Viruses Cells
by a Line of Transformed
CHRISTINE McMILLIN-HELSEL, GEOFFREY M. COOPER
AND
Unrversity of Washington, Department of Medicine, DLuLswn of Oncology, Seattle, Washington 98195; the Fred Hutchinson Cancer Research Center, Seattle, Washington 98104; and the Sidney Farhw Cnncrr Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 Accepted
Ma-v
9, 1978
MSB-1 cells are a line of transformed chicken lymphoid cells derived from tumors induced by Marek’s disease viruses and free of exogenous avian leukosis viruses (ALV). They can be infected by ALV of subgroups A and C including transformation-defective (td) deletion mutants of avian sarcoma viruses (ASV). In terms of virus titers in supcrnatant culture medium, proportion of virus-producing cells, and levels of viral RNA detected by hybridization with a cDNA probe, infection by td ASV of MSB-1 cells was indistinguishable from infection of chicken embryo fibroblasts. In contrast, wild type ASV was restricted in its growth on MSB-1 cells. Different clones of ASV varied in their restriction by all these parameters of viral growth by factors of 10 ’ to 10 4. St,udies of a severely restricted viral clone showed equal quantities of hybridizable viral DNA in Hirt supernatant fractions of both fibroblasts and MSB-1 cells at 10 hr after high multiplicity infection, and transfection assays indicated infectious viral DNA in h&h cell types. Viral DNA largely disappeared from Hirt supernatant fractions of MSB-1 cells by 48 hr after infection, and sarcoma virusspecific DNA was not detected in Hirt pellet fractions from MSB-1 cells at levels found in comparably infected fihroblasts. Infectious ASV DNA, while easily detected in fihroblasts, could not be detected on MSB-1 cells at 48 hr or later times after infection. Because replication of td ASV does not appear restricted in MSB-1 cells, the failure of ASV DNA to integrate normally in these cells seems to be related to the presence of src sequences in the viral genome. INTRODUCTION
Our present understanding of the replication of exogenous avian leukosis-sarcoma viruses is based, for the most part, on studies employing cultured chicken embryo fibroblasts (CEF). As recently reviewed (Vogt, 1977), such studies have revealed three viral genes in both leukosis and sarcoma viruses involved in replication, gag (internal structural proteins), pal (reverse transcriptase), and env (envelope glycoproteins). These are the only known gene functions in field leukosis viruses, helper viruses found in stocks of defective sarcoma viruses, and transformation-defective (td) deletion mutants of nondefective sarcoma viruses. In contrast, the nondefective sarcoma viruses contain a fourth gene src which confers the ability to transform CEF 0042.SS22/78/08~-0~60$02.00/0 Copyright All rights
0 1978 hy Academic Press, of reproductwn m any form
Inc. reserved.
and a limited number of other cell types. Studies of both conditional and nonconditional mutants have not suggested any effect of src on viral replication in CEF. Analysis of the biology of these viruses in other cell types has begun recently. A line of transformed lymphoblasts called MSB-1 derived from a Marek’s disease virus (MDV) -induced lymphoma ( Akiyama and Kato, 1974) is suitable for such studies. These cells contain the MDV genome (Nazerian and Lee, 1974), although only l-2% of the cells contain virus and form MDV antigens (Akiyama and Kato, 1974). They also seem devoid of expression of avian leukosis virus (ALV) information, being negative in chf and gs antigen tests for viral envelope and internal structural proteins, respectively, and lack evidence of exoge-
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nous ALV DNA sequences in hybridization assays (Akiyama and Kato, 1974; Nazerian et al., submitted for publication). They can, however, be exogenously infected by subgroups A and C ALV. In this study, we examined the properties of viral replication on MSB-1 cells and observed that the replication of avian sarcoma viruses (ASV) of subgroups A and C was markedly restricted in these cells while their corresponding td deletion mutants replicated normally. Further study suggested that, following infection of MSB-1 cells with ASV, infectious proviral sequences were synthesized, but the viral DNA did not integrate in host cell DNA in a normal fashion and was lost from the cell. Inasmuch as these abberations did not affect the replication of td ASV, they seemed to be due specifically to the presence of src. MATERIALS
AND
METHODS
Cells. CEF were cultured from chf embryos of the C/E and C/BE phenotypes obtained from H & N Farms, Redmond, Wash,, according to previously described techniques (Rubin, 1960). Transfection experiments utilized CEF of the same phenotype obtained from SPAFAS, Norwich, Conn. MSB-1 cells were contributed by Dr. Kevin Nazerian of the USDA Regional Poultry Research Station, East Lansing, Mich. They were cultivated in a COa incubator at 37” in RPM1 medium 1640 containing 10% fetal calf serum with 55 pg/ml penicillin, 35 pg/ml streptomycin, and 1 pg/ml amphotericin B (complete RPMI). These cells were seeded at 0.5 x lO’/ml and passaged by centrifugation and resuspension in fresh medium every 2 days when the cell density reached about 4 x lO”/ml. MSB-1 cells also formed colonies in soft agar with an efficiency of up to 50% when the following protocol was used. Hard overlay agar used in focus assays (see below) without dimethyl sulfoxide was used as a basal layer in 60-mm plastic tissue culture plates. About 100 MSB-1 cells per plate were overlaid in 3 ml of a medium containing 0.34% agar and 45% (by volume) complete RPMI, 4% tryptose phosphate broth, 10% fetal calf serum, 40% Ham’s medium F10, 1% 100X vitamin concentrate (GIBCO)
OF
ASV
361
supplemented with (by weight) 0.1% sodium bicarbonate and 0.01% folic acid, Cultures were reoverlayed once at 6 days with 0.45% agar containing (by volume) 50% medium F-10 with 5% fetal calf serum and 50% complete RPMI. Viruses, ASV selected for study were Prague strain of Rous sarcoma virus of subgroups A and C (PR-A, PR-C) and the subgroup C virus, B-77. All sarcoma viruses were cloned from either foci or agar colonies. Among the PR-A viruses, clones lb, lc, 5, and 6 were produced by cells grown from individual foci of transformation while clones 2a, 4c, 5b, and 6b represented viruses produced by cells from agar colonies of CEF transformed by stock PR-A. Similarly, B77 clone 1 and PR-C clones 4a and lc were derived from foci, whereas PR-C, 4a clones 3, 11, 14, and 20 were all subclones of PR-C clone 4a derived from agar colonies. Leukosis viruses studied were td RSV, laboratory stocks of td PR-C and td PR-A, and newly isolated td PR-C lc recovered by endpoint dilution of parental PR-C clone lc grown on MSB-1 cells (as described in Resuits). Virus infection and assays. About 1 x 10” MSB-1 cells were suspended in 0.5 ml F-10 growth medium and infected with the appropriate dilution of virus for 20 min at 37”. For subgroup C viruses, 2 pg/ml polybrene (Aldrich) was present. The cells were then centrifuged, resuspended in RPM1 growth medium and cultured as described above. Supernatant culture medium derived from MSB-1 or CEF was assayed by focus assays for ASV and by the endpoint interference method for td RSV as previously described (Wyke and Linial, 1973; Linial and Neiman, 1974). For measuring the capacity of infected MSB-1 cells to form infectious centers, the following protocol was used. An appropriate number of infected cells were suspended in F-10 growth medium at a concentration of l-5 x lo”/ml and irradiated with 840 R from a “‘7Cs source. At this dose, all MSB-1 cells were killed. The irradiated cell suspension was then serially diluted and plated on freshly prepared monolayers of CEF. For ASV-infected MSB-1 cells, these monolayers were then overlayed 24 hr later and examined
362
NEIMAN
for foci of transformation in the standard fashion. The proportion of infective centers among the MSB-1 cells was estimated from the dilution and the number of foci counted. For td RSV-infected MSB-1 cells, plates of CEF innoculated with irradiated MSB-1 cells were passaged three times to ensure complete infection and then challenged with ASV of the same subgroup. The proportion of infective centers among the MSB-1 cells was then calculated from the dilution of MSB-1 cells at which interference was no longer detected. Assays of reverse transcriptase in culture supernatants. Concentration of virus particles released in cultures of infected MSB-1 cells and CEF was also estimated by measurement of reverse transcriptase levels in supernatant medium. Aliquots, 3-10 ml, of tissue culture fluid were clarified at 10,000 g for 10 min and virus sedimented from supernatant at 150,000 g for 30 min. Viral pellets were dissolved in reaction mixtures containing 50 mM Tris, pH 8.3, 1% NP-40, 10 mM dithiothreitol, 6 mM Mg(C&02)2, 60 mM NaCl, and 0.02 mM each of d ATP, d CTP, d GTP, and [“HITTP (50 Ci/mM). Reactions were stopped by the addition of lo%> trichloroacetic acid (TCA) and DNA synthesis assayed as cpm incorporated into TCA-precipitable material. The rate of reaction was assessed as PM of TTP incorporated/hr/ml of tissue culture fluid from the linear portion of the reaction curve (generally, 15 and 30-min samples showing a I:2 ratio of incorporated cpm). Control reactions for background determinations contained “viral pellets” from uninfected tissue culture fluid. Preparation and labeling of viral RNA and cDNA. Viral 70 S complex RNA was isolated from PR-C and labeled to a specific activity of 2-4 X 1Oi cpm as previously described (Neiman et al., 1974). The preparation and characterization of ‘“‘I-labeled fragments of the RSV genome composed of the 2000 nucleotides at the 3’-terminus is described elsewhere (Neiman et al., 1977). Single-stranded cDNA complementary to PR-RSV RNA was prepared as follows. PR-C 35s RNA, 1 pg [prepared as described previously (Neiman et al., 1977, 1978)], was incubated for 4 hr at 37” in a 50-~1 reaction
ET
AL.
mixture of 0.5 M Tris, pH 8.3, 0.18 M NaCl, 0.6 mu Mg(C2H302)2, 10 mM dithiothreitol, 0.125 pg/ml actinomycin D, 0.5 mM each of dATP, dCTP, and dGTP, 0.1 mM [“H]TTP (50 Ci/mmol) with 100 pg of DNase I limit digest calf thymus DNA fragments as primer (Taylor et al., 1976) and 20 units of AMV reverse transcriptase (a gift of Dr. Joseph Beard, 1 unit of enzyme incorporates 100 pM of TTP in 15 min). Reaction mixtures containing the digested DNA primer, but lacking template RNA, incorporated 0.002% of the quantity of acid-precipitable [“HIDNA product obtained in complete reaction mixtures. Reaction mixtures were diluted in 0.25 ml of 1 M NaCl, 0.1 M Tris, 0.01 M EDTA, pH 7.2, and extracted with an equal volume of chloroform-phenol mixture (l:l, v/v). The aqueous phase was diluted to 2 ml in 0.1 N NaOH and incubated in the dark at 20” for 16 hr. The solution was then neutralized and the DNA precipitated with 2 volumes of ethanol at -20”. The cDNA was dissolved in 20 ~1 5X SSC, 50% formamide containing 1 pg of RSV RNA and incubated for 24 hr. The reaction mixture was diluted in 5 ml of a solution of CsSOd in 2 mM EDTA with a density of 1.54 g/liter, centrifuged at 180,000 g for 60 hr, the gradient fractions in the region of hybrid density (1.48-1.62 g/liter) pooled and dialyzed against 0.01X SSC, the RNA hydrolyzed by treatment with 0.1 N NaOH, and finally the single-stranded cDNA product precipitated with 2 volumes of ethanol. The [“H]cDNA probe prepared in this fashion had a specific activity of about 2 X lo7 cpm/pg and an S1 nuclease sensitivity of 94%. When reacted with ““I-labeled RSV RNA in 5X SSC, 50% formamide at 50” for 24 hr (Cot value for cDNA = 2.7 X 10’) at cDNA:[““I]RNA mass ratios of 2:l and l:l, 22%) and 16%, respectively, of the viral RNA became resistant to pancreatic RNase. Conversely, about 75% of the cDNA probe was rendered resistant to S1 nuclease by hybridization with an excess of viral RNA. Preparation of cellular DNA. Fibroblasts or MSB-1 cells (2 X lO’/ml) were suspended in 0.1 M Tris, 0.001 M EDTA (T.E.) and lysed at 37” in the presence of 1% SDS. DNA was fractionated by the
RESTRICTION
method of Hirt (1967) by making the mixture 1 M NaCl and holding at 4” for 8-16 hr. The pellet fraction (“Hirt pellet”), obtained by centrifugation at 17,000 g for 15 min, was redissolved in T.E., and the DNA extracted with multiple spoolings on a glass rod by the method of Marmur (1971). The supernatant fraction (“Hirt supernatant”) was extracted with an equal volume of phenol/chloroform l:l, v/v) and precipitated with 2 volumes of cold ethanol. The pellet was redissolved in T.E. containing 0.1 M NaCl (standard buffer) and digested with 50 pg/ml pancreatic ribonuclease for 30 min at 37” followed by 50 pg/ml of pronase for 2 hr. The solution was then reextracted with an equal volume of phenol/chloroform and DNA precipitated from the aqueous phase with 2 volumes of cold ethanol. Preparation of cellular RNA. Recovery of RNA from MSB-1 cells was very poor in our hands using standard phenol-SDS or protease methods. Reasonable recoveries were obtained using the recently described guanidine extraction method (Strohman et al., 1977; Adams et al., 1977). MSB-1 cells (1 X 10” cells/ml) were suspended in 8 M guanidine-HCl, 10 mM Na(C2H302), pH 5.2, 1 mM dithiothreitol in a Dounce homogenizer and homogenized with 10 strokes of the “B” pestle. One-half volume of ethanol was added, the mixture held at -20’ for 30 min, and the RNA-containing material pelleted at 10,000 g for 10 min. The pellet was redissolved in 8 M guanidine-HCl, 10 mM Na(CnH:rOs), 20 mM EDTA, pH 5.2 (0.25 volume of the original homogenate), and reprecipitated with 0.5 volume of cold ethanol. This procedure was repeated three times. The final pellet was then dissolved in 20 mM EDTA, pH 7.0, and extracted with equal volumes of a 4:l chloroform/butanol mixture. The interface and organic phase were re-extracted in 0.5 volume of the EDTA solution and the two aqueous phases combined. This solution was made 3 M in Na(C&O.J, pH 5.2, and the RNA precipitated overnight in -20”. The RNA pellet was finally redissolved in 0.1 M Na( C,H,,O,), pH 5.2, and reprecipitated with ethanol. Recoveries were on the order of l-2 mg of RNA/5 X 10” MSB-1 cells which was dissolved in water and stored in liquid nitrogen
OF
363
ASV
until use. Hybridization conditions. All hybridization reactions with either [3H]cDNA or [1251]RNA probes were carried out in 0.75 M NaCl, 0.75 M C6H5Na307, 50% formamide solution at 50”, and in all cases, viral sequences in cellular DNA or RNA were present in excess. Hybrids were assayed as previously described by resistance to S1 nuclease when [“H]cDNA was used as a probe (Leong et al., 1972) and by pancreatic RNase when [‘““IIRNA was used as a probe (Neiman et al., 1974). The extent of reaction was measured as a function of CRot or Cot (mol-set/liter) based on either viral RNA or bulk cellular RNA or DNA concentrations when viral RNA or cellular RNA or Hirt pellet DNA was used as “driver.” When Hirt supernatant DNA was used as driver, hybridization was plotted as a function of C&t based on the number of cell equivalents present in the reaction mixture. The number of cells used for extraction was divided by the volume of the final DNA solution to obtain the number of cells represented per unit volume (cell equivalent, CE). The number of CE/pl in each reaction mixture was multiplied by the reaction time in seconds to obtain a value of CEot for each time point in the reaction. Individual reaction mixtures had concentrations of 1-3 x 10” CE/pl. Transfection assays. DNA from RSV-infected fibroblasts and MSB-1 cells were assayed for infectivity using the calcium method of Graham and Van der Eb (1973) as previously described (Cooper and Temin, 1976). Recipient cultures of CEF were exposed to 5 pg of total cell or Hirt pellet DNA preparations, or to approximately 1 X 10’ cell equivalents of Hirt supernatant DNAs in the presence of 5 pg of salmon sperm DNA. Foci of transformed cells were scored on the original DNAtreated plates. In addition, all recipient. cultures were tested for production of RSV either by transferring the cells or by assaying a sample of the supernatant medium on fresh CEF. RESULTS
Growth of ASV and td ASV on MSB-1 cells. MSB-1 cells were plated in soft agar
364
NEIMAN
and six clones were picked and established in culture, ASV and td ASV were used to infect these cells at a multiplicity of infection (m.o.i.) [based on focus-forming units (FFU) or interference units (IU) titered on CEF] of 0.03 and the supernatant culture fluids harvested at various times and assayed for FFU and/or IU on CEF and, in some cases, for reverse transcriptase levels. Figure 1 shows the growth of two agar derived clones of PR-A on six different clones of MSB-1 cells. Between 3 and 7 days after infection virus titers reach maximum levels which clustered for all MSB-1 cell clones around 10” FFU/ml (range, 104-10”) following infection with PR-A clone 2A and around 10” FFU/ml following infection with PR-A clone 6B. An exception was the somewhat higher titer (about lo4 FFU/ml) seen on clone IV MSB-1 cells. However, given the relatively uniform growth of a given ASV on the different cell clones, a single one, MSB-1 clone II, was selected for further analysis. Table 1 summarizes the titers of a panel of different ASV of subgroups A and C grown on MSB-1 clone II cells. A wide range of titers was observed with different virus clones ranging from very low levels to those approaching titers achieved on CEF. Where released virus particles were mea-
FIG. 1. Replication of RSV on different clones of MSB-I cells. MSB-1 cell stock was plated in soft agar as described in Materials and Methods and six clones; I, 0; II, q III, a; IV, A; V, 0; VI, n were picked, cultured, and infected with either PR-A clone 2a (panel A) or PR-A clone 6b (panel B) at an m.o.i. of 0.03. The infected cells were cultured in 2 ml of medium in X-mm plastic tissue culture dishes and the medium changed at 2.day intervals. The titer of FFU/ml in the supernatant medium was assayed and normalized for the number of cells in the culture at the time of assay.
ET
AL
sured by reverse transcriptase levels, virus production from MSB-1 cells seemed also reduced by a factor of lo”-lo4 as compared to that achieved on fibroblasts although the stoichiometry between enzyme levels and biological particles was, for unclear reasons, not precise. In contrast, virus titers from td RSV-infected MSB- 1 cells were uniformly near those achieved on fibroblasts. Thus, there appeared to be a restriction to replication of ASV on MSB-1 cells which varied from virus clone to virus clone and was either less apparent or did not apply at all to td deletion mutants of ASV. One consequence of this observation was that it was sometimes easy to recover td RSV from stocks of focus-cloned RSV simply by growth on MSB-1 cells presumably because low levels of such mutants in the sarcoma virus stock could replicate to high titer while the parental RSV remained restricted. For example, td PR-C lc was recovered by dilution of supernatant medium following infection of MSB-1 cells with the parental PR-C clone lc stock. As shown in Table 1, this virus replicated to about the same levels as stock td RSV in both fibroblasts and MSB-1 cells. Attempts to recover td RSV from freshly agar-cloned RSV by simple endpoint dilution were not successful. Effect of virus multiplicity on replication in MSB-1 cells. In this experiment, PR-C clone lc and td PR-C lc were compared. MSB-1 clone II cells were infected with either virus at various multiplicities. Six days after infection, supernatant medium was harvested for measurement of virus titers and appropriate dilutions of irradiated cells were plated on CEF for measurement of infectious centers. Figure 2 shows that for td RSV, the proportion of cells scoring as infectious centers increased linearly with increasing m.o.i. until the majority of cells yielded virus at an m.o.i. of about 0.1 (Fig. 2B). The titer of IU in the supernatant medium increased in proportion to the fraction of infectious centers (Fig. 2A). For the sarcoma virus, the proportion of infectious centers also increased linearly with m.o.i., but remained reduced lo:‘- to 104-fold in comparison with td RSV infection. Focus-forming activity in the super-
RESTRICTION
OF
TABLE VIRAL
Titer”
Virus
_ .___ -.. ~-~--.--~ Focus clones, subgroup PR-A clone lb PR-A clone 1 c PR-A clone 5 PR-A clone 6
2 x 1oi 4 x lOA >2 x 10’ 5 x 10’
A
Focus
C
clones
subgroup
RSV(RAV-7).2 B-77 clone 1 PR-C clone 4a
Reverse transcrip ~...~~ tase
A
Agar clones, subgroup PR-A clone 2a PR-A clone 4c I’R-A clone 5b I’R-A clone 6b
RSV(RAV-71-l
GROWTH
4.5 6.1 7.5 1.4
1
ON MSB-
x x x x
10” 10’ 10’ IO’
1.3 1.5 1 1
x x x x
10’ IO4 IO’ IO’
I CELLS’< .I‘i(er”
Virus
~~~~--Agar reclones PIT-(’ PR-C-4a-3 I’R-C-4a-11 PR-C-4a14 I’K-C-4a-20 Leukosis
8 3 4 1
365
ASV
td td trl
0.11 0.12
All
clone
4d’
7 x IO’
0.67
4 x 10’ 1 x IO’ 2 x IO
2.44 03; 1.74
viruses
I’R-A PR-C I’R-C-lc
viruses
Reverse transcrip. t ---asra’
5 x 10” 1 x 10” H x 10’ groum
on fibrobln.stL
0.42
” All infections were of MSB-1 clone II cells at an m.o.i. of 0.01 to 0.1. Assays were 5-7 days after infection. ” Titer FFU/ml for ASV or IU/ml for td ASV [for RSV (RAV-7), FFlJ/ml were measured]. ’ L’HlTTP incorporation in pmot/hr/ml of medium. ” PR-C clone 4a-infected fibroblasts were plated in soft agar, colonies picked and cult,ured. and supernatant virus from four colonies reassayed on MSB-1 clone II cells. ” Value from I’R-C-lc-infected CEF cultures.
natant medium also varied in proportion to the fraction of infectious centers about lO:‘fold below the titer achieved with td RSV. These data suggested that the small amount of RSV produced following infection of MSB-1 cells comes from a limited proportion of cells rather than from low level production by the majority of cells. Further, the linear relationship with m.o.i. up to a limit suggested the titration of a small fraction of either virus or MSB-1 cells capable of productive infection. If there existed a small proportion of virus infectious for MSB-1 cells in the RSV stock (maximally, 1 in 10:’ CEF infectious particles) they should have been easily eliminated by recloning the virus stock. As shown in Table 1, however, recloning of this virus by the agar colony technique did not eliminate the low level of RSV production. lJnless this focus-forming activity represented variants rapidly accumulating in the virus stock after cloning, the most reasonable interpretation seemed to be that RSV was produced by a small proportion of sus-
ceptible cells (far smaller than occurred with td RSV infection) which could be increased, somewhat, by increasing m.o.i. This idea was tested further by an experiment in which MSB-1 clone II cells were plated in soft agar (to reduce viral spread) immediately after infection at an m.o.i. of 2 with PR-C clone 4a. As might be expected from the experiment shown in Fig. 2, none of 10 resulting presumptively infected MSB-1 clones produced virus by assay of FFU or reverse transcriptase (data not shown). Cells from these clones could reasonably be assumed to represent the consequences of nonproductive infection of MSB-1 cells by RSV and were used along with cells from infected and uninfected masscultures, for further biochemical analysis. Viral RNA levels in MSB-I cells. Viral RNA levels in MSB-1 clone II cells were assessedbefore and after viral infection by hybridization with a [“H]cDNA to PR-RSV RNA. The results of that analysis are shown in Fig. 3 in which the extent of
366
NEIMAN
I
FIG. 2. Effect of multiplicity of infection. MSB-1 clone II cells were infected with either PR-C clone lc, 0, or td PR-C clone IC, 0, at various m.o.i., and the cells cultured for 6 days. Panel A shows the titer of IU, 0, or FFU/ml, 0, in the supernatant medium, and panel B shows the proportion of infective centers for the two viruses as a function of the initial m.o.i.
hybridization of the probe is plotted as a function of CRot based on the concentration of total cellular RNA in the reaction mixture. Under the conditions of these hybridization assays, the CRotlp observed with PR-RSV template RNA was about 8 x lo-“, whereas that of RNA from td RSVinfected MSB-1 cells was about 2 X 10” suggesting that about 0.4% of RNA from td RSV-infected cells was complementary to the viral cDNA probe. The slightly lower extent of hybridization in comparison to that seen with template RNA may be attributable to the absence of src-related RNA sequences in the td RSV-infected cells. This level of viral RNA is comparable to that reported for infected CEF using similar methodologies (Leong et al., 1972; Coffin and Temin, 1972; Hayward, 1977). In this connection, the figure demonstrates that we detected with this cDNA more viral RNA in uninfected MSB-1 cells (which are functionally gs- chf) than has been ob-
ET AL served in uninfected CEF with the same level of endogenous viral antigen expression (Hayward and Hanafusa, 1973; Hayward, 1977). Nevertheless, this RNA concentration is still (at a CRotlp of about 4 x 103) lower than that observed in gs+ chf+ fibroblast cultures. To the point of these experiments, Fig. 2 demonstrates that RNA from MSB-1 clone II cells infected at an m.o.i. of 2 and then recloned immediately in soft agar (see preceding section) contained no more RNA detectable with our cDNA probe than did uninfected cells. Cultures of MSB-1 clone II cells which were infected with RSV at the same high m.o.i. and grown for a week in standard culture contained a level of viral RNA roughly proportional to the titer of FFU in the supernatant at the time of extraction. For example, Fig. 2 shows an analysis of such cells infected with PR-C clone 4a at a point when the FFU titer in the culture was about 2 x 104/ml. The CRotlp observed was about lo”, corresponding roughly to a 50- to loo-fold lower viral RNA concentration that observed in td RSV infected cells. Thus, within the limits of the viral sequence representation of the cDNA, and the sensitivity of the method, we detected no clear evidence of transcription of exogenous viral genes in
FIG. 3. Concentration of viral RNA in infected MSB-1 clone II cells. About 1000 cpm (0.05 ng) of the cDNA probe to PR-RSV RNA was incubated for various time periods with either 1 ng RSV RNA, q 1 fig total cell RNA from td PR-C-infected MSB-1 cells, 0; 25 lrg total cell RNA from PR-C-4a subclone 3infected MSB-1 cells, 0; 50 g total cell RNA from PR-C-4a-infected and recloned MSB-1 clone II cells (see text), a; or 50 /~g total cell RNA from uninfected MSB-1 cells, W, in 5 ~150% formamide, 5X SSC at SO”. Hybridization was plotted as a function of CK,t (molset/liter) where CR,, = concentration of RNA nucleotides in mol/liter and t is the time in seconds.
RESTRICTION
the vast majority of RSV-infected MSB-1 cells. Detection of viral DNA in Hirt supernatant fraction. Inasmuch as we could detect no clear evidence of either the formation and release of virus particles or the accumulation of viral RNA in the large majority of MSB-1 cells following infection with ASV, it seemed reasonable to examine the early events after virus infection for evidence suggesting the mechanism of restriction of sarcoma virus replication. DNA preparations from Hirt supernatant fractions extracted from MSB-1 clone II cells and CEF 10 hr following infection with PRC clone 4a at an m.o.i. of 2 were analyzed by hybridization with the RSV cDNA probe. The results are shown in Fig. 4. As described in Materials and Methods, the data are compared on the basis of the number of cell equivalents contributing to the DNA in each reaction mixture. On this basis, the cDNA probe detected an apparently nearly equal concentration of complementary viral DNA sequences in Hirt su-
log
CE,t
FIG. 4. Viral DNA sequences in Hirt supernatant DNA. DNA from a portion of the Hirt supernatant fraction representing either 5 X 10” MSB-I clone II cells, * or CEF, 0; 10 hr after infection, or 8 x 10’ MS&l clone II cells 48 hr after infection with PR-C clone 4a at an m.o.i. of 2 was incubated with 1000 cpm of the PR-RSV cDNA probe under the same conditions as for Fig. 3. Hybridization is plotted as a function of C&t where CE,, = cell equivalents represented by the Hirt supernatant extract/PI of hybridization reaction mixture and t = time in seconds.
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367
pernatant fractions of both CEF and MSB1 cells 10 hr following infection. In contrast, the Hirt supernatant fraction from MSB-1 cells 48 hr after infection with the sarcoma virus contained very little DNA detectable with the cDNA probe. Using a more complex ‘““I-labeled viral genomic RNA probe, virtually no sequences were detected above background in this fraction (data not shown). Other investigators have reported a high concentration of apparently unintegrated proviral sequences in infected CEF persisting up to 3 days after infection (Khoury and Hanafusa, 1976), but, in MSB1 cells, we thus observed that the initially synthesized proviral sequences have largely disappeared by 48 hr after infection. Detection of viral DNA in Hirt pellet fractions. DNA was extracted from Hirt pellet fractions of MSB-1 cells 2 days and 16 days after infection with PR-C clone 4a at an m.o.i. of 2. In the latter case, clone II cells were recloned in soft agar immediately after infection, and clones were picked and cultured for 16 days to obtain an adequate number of cells for extraction as described before. A Hirt pellet fraction was also prepared 18 days after infection at an m.o.i. of 10. Because of the limited sequence representation in the cDNA probe, and the presence of endogenous RSV-related sequences in the normal chicken genome, an ‘““I-labeled genomic RNA probe was used which clearly distinguished between the DNA of infected and uninfected cells with respect to viral sequences (Neiman, 1972; Neiman et al., 19’74). For purposes of comparison, DNA from PR-C-transformed CEF and uninfected chick embryo cells was also analyzed. The kinetics of hybridization reactions between the lZ51viral RNA probe and these various DNA preparations are shown in Fig. 5. None of the MSB-1 cell DNA preparations formed hybrids with viral RNA with a rate or extent comparable with that observed with DNA from infected CEF (previously estimated to contain 2-4 proviruses/haploid genome) (Neiman, 1972; Wright and Neiman, 1974; Neiman et a!., 1974). The MSB-1 cells infected at an m.o.i. of 2 seemed to have no more reactive DNA than the uninfected cells. More hybridization was detected with MSB-1 cell DNA
368
NEIMAN
I
IO’
I03
104
105
co+
FIG. 5. Viral DNA sequences in Hirt pellet DNA. Panel A shows the kinetics of hybridization of 2oOO cpm ‘?-labeled PR-RSV RNA with 200 pg of cellular DNA from I’R-C-transformed fibroblasts, 0; uninfected CEF, 0; MSB-I clone II cells infected with PRC clone 4a at an m.o.i. of 2 48 hr after infection, e 16 days after infection and recloning, A; and 18 days after infection with the same virus at an m.o.i. of 10, & in 20 ~1 of hybridization mixture under the same condtions as described in the preceding figures. Panel B shows hybridization of “‘I-labeled 3’ end fragments of PR-C subunit RNA representing the terminal 2000 nucleotides of the viral genome with the MSB-1 Hirt pellet DNA from the m.o.i. = 10 infection used in panel A, m and from MSB-I cells infected with I’R-C clone 4a at an m.o.i. of 0.3, v. Conditions were as for panel A.
after infection at an m.o.i. of 10. To determine whether this increased hybridization reflected exogenously introduced sarcoma virus DNA sequences, a special ‘““I-labeled PR-C probe was prepared composed of sequences comprising the 2000 nucleotides at the 3’ end of the viral genome. This segment includes about 1500 nucleotides of the src gene of the virus and a terminal region shared with td RSV (Wang et al., 1975; Coffin and Billeter, 1976; Junghans et al., 1977; Neiman et al., 1977). In the presence of an adequate concentration of unlabeled competitor RNA from td RSV, and under these conditions of hybridization, the maRSV sequence jority of this ‘““I-labeled
ET
AL.
forms hybrids with DNA from RSV-infected CEF, whereas very little forms hybrids with normal chicken DNA as previously reported in detail (Neiman et al., 1977). This fragment could be used to attempt to detect RSV-specific sequences in MSB-1 cell DNA. Figure 5B shows that DNA from the MSB-1 cells infected at an m.o.i. of 10 formed hybrids with this srcspecific probe, but very little reaction was detected in MSB-1 cells infected at a lower multiplicity (0.3). The possibility exists that these sequences found in Hirt pellet DNA might be nonspecifically trapped, unintegrated viral DNA. This explanation seems, however, extremely unlikely since we have detected very little or no viral DNA in Hirt supernatant fractions at the intervals after infection when these analyses were performed (see Fig. 4). For example, the 3’ end fragment used as a src-specific probe formed no hybrids at all with Hirt supernatant fraction DNA obtained from the MSB-1 cells infected at an m.o.i. of 10 (data not shown). Thus, some RSV-specific sequences detected probably do either integrate or at least “associate” with chromosomal DNA in a manner leading to precipitation by the Hirt procedure. Nevertheless, the process did not occur as extensively in most RSV-infected MSB-1 cells as occurs in RSV-infected CEF, and appeared to require an extremely high m.o.i. Finally, the data obtained in this analysis of both Hirt supernatant and pellet fractions did not indicate whether complete or otherwise “normal” proviruses are found in most MSB-1 cells following infection with RSV. Formation of infectious DNA after infection with RSV. The infectivity of DNA extracted from PR-C-infected MSB-1 cells was assayed by transfection to determine whether complete copies of RSV DNA were present (Table 2). Hirt supernatant DNAs extracted 10 hr after infection of both CEF and MSB-1 clone II cells with PR-C clone 4a (m.o.i. = 2) contained infectious RSV DNA, although the DNA of PR-C-infected MSB-1 cells appeared less infectious than the DNA of PR-C-infected CEF. PR-C-infected CEF also contained infectious RSV DNA 48 hr after infection and 14 days after infection. In contrast, no infectious RSV
RESTRICTION TABLE INFECTIVITY
OF RSV
FIBROBLASTS
Donor cells CEF
MSB-1
DNA
2
DNA AND
OF PR-C-INFECTED
MSB-I
Hirt supernatant, Total cell, 48 hai Total cell, 14 dai
CELLS~’
Fraction positive recipient cultures’
preparationh
10 hai
Hirt supernatant, 10 hai Hirt pellet, 48 hai Total cell, 48 hai Hirt pellet, 18 dai
6/16
W3 9/12 2/lfi O/5 o/9 O/8
” Recipient cultures of CEF were treated with 5 ag of total cell or Hirt pellet DNA preparations, or with approximately 1 x 1Oi cell equivalents of Hirt supernatant DNA. Transformation was scored on the DNAtreated plates and supernatant media harvested 7 days after DNA treatment was assayed on fresh CEF to test for production of RSV. * DNAs were extracted at the indicated times after infection (hai, hours after infection; dai, days after infection) of CEF or MSB-1 clone II cells with PR-C clone 4a at an m.o.i. of 2, except for the Hirt pellet DNA extracted 18 days after infection of MSB cells at an m.0.i. of 10. ’ Number of DNA-treated cultures that produced progeny RSV over the total number tested.
DNA was detected 48 hr after infection of MSB-1 cells with PR-C. In addition, no infectivity was detected in assays of the Hirt pellet DNA of MSB-1 cells infected with PR-C at an m.o.i. of 10, although this DNA contained PR-C-specific DNA sequences detected by hybridization (Fig. 5). These results, therefore, indicated that infectious RSV DNA was synthesized in PRC-infected MSB-1 cells, but was present only at early times after infection. DISCUSSION
The principal conclusion from this set of observations is that the replication of ASV in most MSB-1 cells is restricted in comparison to their replication on fibroblasts. The data suggest that in the majority of MSB-1 cells, infection with ASV fails to result in the production of virus particles although in a small fraction of infected cells there appears to be production of focus forming virus. The fraction of cells capable of productive infection varies with different
OF
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369
clones of ASV, and can be increased somewhat by high multiplicities of infection. In contrast, td deletion mutants of RSV replicate, in terms of virus titers, infectious centers, and intracellular concentrations of viral RNA to roughly equivalent levels on both MSB-1 cells and CEF strongly suggesting that the presence of src sequences in the ASV genome is critical for the operation of the MSB-1 cell restriction. It should be pointed out that these conclusions apply to viruses of subgroups A and C. Neither leukosis nor sarcoma viruses of other subgroups (e.g., B, D, E) will replicate in MSB-1 cells, presumably on the basis of a surface barrier. Our studies of the event in the virus life cycle where ASV replication is interrupted indicated that the restriction occurs as a result of a block in events occurring early after infection. Hybridization and transfection analyses suggested that presumptively unintegrated viral DNA sequences accumulated in apparently normal quantities by 10 hr after infection, and that this viral DNA is infectious. By 48 hr after infection, most of this free DNA is no longer detected in Hirt supernatant fractions, and, in contrast to the situation in ASV-infected fibroblasts, infectious DNA can no longer be detected in total cell DNA. It thus appears that while normal ASV DNA is initially formed in MSB-1 cells, this viral DNA is lost from the cell. Some ASV-specific sequences do become either closely associated with or integrated into chromosomal DNA in a large fraction of MSB-1 cells after infection at very high m.o.i. We did not obtain evidence, however, that these sequences represented infectious proviral DNA. The restriction of ASV replication on MSB-1 cells bears some resemblance to the Fv-1 restriction of murine leukemia virus replication, which appears to act before integration of viral DNA, but after synthesis of virus-specific DNA detectable by nucleic acid hybridization (Jolicoeur and Baltimore, 1976; Sveda and Soeiro, 1976). A mechanism is needed to explain how MSB-1 cells discriminate between the newly formed provirus of ASV and that of td ASV. The presence of a nuclease in MSB-1 cells (perhaps analogous to bacte-
370
NEIMAN
rial restriction endonuclease) which recognizes specific src DNA sequences is an attractive hypothesis which is compatible with all of the observations we have made and which may be testable. We have not as yet obtained any direct evidence of specific degradation of ASV DNA. Whatever the specific factors are that obstruct replication of ASV, it is clear that they are far more evident in MSB-1 cells than in CEF. It is possible that the mechanism for restricting ASV is simply absent in CEF. However, considerable variation was seen in the efficiency of restriction of different clones of subgroup A and C ASV by MSB-1 cells (see Table 1). Because ASV have been selected for growth on CEF for many years, these agents may have changed their src sequences sufficiently to overcome restriction by these cells. That ASV provirus integration occurs more readily in CEF than this lymphoid cell line may indicate at least part of the reason for the apparent specificity of fibroblasts and related cell types for in vivo transformation by ASV. We hasten to add, however, that in addition to being lymphoid, MSB-1 cells differ from CEF in being both transformed and infected with MDV. Which of these factors is significant in determining the specific restriction of ASV in MSB-1 cells remains to be established. ACKNOWLEDGMENTS We thank Don Macdonnell and Sharon Okenquist for excellent technical assistance. This investigation was supported by Grants CA 20068, CA 15704, and CA 18689, awarded by the National Cancer Institute. Dr. Neiman is a Scholar of the Leukemia Society of America. REFERENCES S. L., SOBEL, M. E., HOWARD, B. H., OLDEN, K., YAMADA, K. M., DE CROMBRUGGHE, B., and PASTEN, I. (1977). Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Nut. Acad. Sci. USA 74, 3399-3403. AKIYAMA, Y., and KATO, S. (1974). Two cell lines from lymphomas of Marek’s disease. B&en J. 17, 105-116. COFFIN, J. M., and TEMIN, H. M. (1972). Hybridization of Rous sarcoma virus DNA polymerase product and ribonucleic acids from chicken and rat cells infected with Rous sarcoma virus. J. Viral. 9, ADAMS,
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AL.
766-775. COFFIN, J. M., and BILLETER, M. A. (1976). A physical map of the Rous sarcoma virus genome. J. Mol. Biol. 100, 293-318. COOPER, G. M., and TEMIN, H. M. (1976). Lack of infectivity of the endogenous avian leukosis virusrelated genes in the DNA of uninfected chicken cells. c1. Vmll. 17, 422-430. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,456-467. HAYWARD, W. S., and HANAFUSA, H. (1973). Detection of avian tumor virus RNA in uninfected chicken embryo cells. J. Viral. 11, 157-167. HAYWARD, W. S. (1977). Size and genetic content of viral RNAs in avian oncovirus-infected cells. J. Virol. 24,47-63. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369. JOLICOEUR, P., and BALTIMORE, D. (1976). Effect of Fv-I gene product on proviral DNA formation and integration in cells infected with murine leukemia viruses. Proc. Nat. Acad. Sci. USA 73, 2236-2240. JUNGHANS, R. P., Hu, S., KNIGHT, C. A., and DAVIDSON, N. (1976). Heteroduplex analysis of avian RNA tumor viruses. Proc. Nat. Acad. Sci. USA 74, 477-481. KHOURY, A. I., and HANAFUSA, H. (1976). Synthesis and integration of viral DNA at different times after infection with various multiplicities of avian oncoviruses. J. Virol. 18, 383-400. LEONG, J. A., GARAPIN, A., JACKSON, N., FANSHIER, L., LEVINSON, W. E., and BISHOP, J. M. (1972). Virus-specific ribonucleic acid in cells producing Rous sarcoma virus: Detection and characterization. J. Viral. 9, 891-902. LINIAL, M.. and NEIMAN, P. (1976). Infection of chick cells by subgroup E viruses. Virology 73, 508-520. MARMUR, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3, 208-223. NAZERIAN, K., and LEE, L. F. (1974). Deoxyribonucleic acid of Marek’s disease virus in a lymphoblastoid cell line from Marek’s disease tumour. J. Gen. Virol. 25, 317-321. NEIMAN, P. E. (1972). Rous sarcoma virus nucleotide sequences in cellular DNA: Measurement of RNADNA hybridization. Science 178, 750-753. NEIMAN, P. E., WRIGHT, S. E., MCMILLIN, C., and MACDONNELL, D. (1974). Nucleotide sequence relationships of avian RNA tumor viruses: Measurement of the deletion in a transformation-defective mutant of Rous sarcoma virus. J. Viral. 13,837-846. NEIMAN, P. E., DAS, S., MACDONNELL, D., and MCMILLIN-HELSEL, C. (1977). Organization of shared and unshared sequences in the genomes of chicken endogenous and sarcoma viruses. Cell 11, 321-329.
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265-273.
“Comprehensive Virology,” Vol. IX. pp. 34 I-455 (H. Fraenkel-Conrat and Ii. Wagner, eds.). Plenum Press, New York. WANG, L. H., DUESBERG, l’., BEEMON, K., and VOGT, 1~. K. (1975). Mapping T,-resistant oligonucleotides of avian tumor virus RNAs: Sarcoma specific oligonucleotides are near the poly(A) end and oligonucleotides common to sarcoma and transformation defective viruses are at the poly(A) end. ,J. Viral.
M. M., and SOEIRO, R. (1976) Host restriction of Friend leukemia virus: Synthesis and integration of the provirus. Proc. Nat. Acad. Sci. USA 73,
WRIGHT, quence
H. (1960). A virus in chick embryos which induces resistance in uiko to infection with Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 46, 1105-1119. STROHMAN, R. C., Moss, P. S., MICOU-EASTWOOD, J., SPECTOR, D., PRZYBYLA, A., and PATERSON, B. (1977). Messenger RNA for myosin polypeptides: Isolation from single myogenic cell cultures. Cell 10, KUBIN,
SVEDA,
2356-2360. It., and SUMMERS, .J. (1976). Efficient transcription of RNA into DNA by avian sarcoma virus polymerase. Biochim. Biophys. AIfrr 442, 324-330. VOG~, P. K. (1977). Genetics of RNA tumor viruses. TAYLOR,
.J. M.,
ILLMENSEE,
In
16,
1051-1070.
S. E., and relationships
NEIMAN,
between
P. E. (1974). Base-seavian ribonucleic acid
endogenous and sarcoma viruses assayed petitive ribonucleic acid-deoxyribonucleic bridization. Biochemistry 13, 1549-1554. WYKE, tive
J., and
LINIAL,
avian sarcoma terization of twenty
by comacid hy-
M. (1973). Temperature-sensiviruses: A physiological characmutants. Virology 53, 152-361.
VIROLOGY
Specific
(1978)
Restriction
PAUL
of Avian
E. NEIMAN,
Sarcoma Lymphoid
Viruses Cells
by a Line of Transformed
CHRISTINE McMILLIN-HELSEL, GEOFFREY M. COOPER
AND
Unrversity of Washington, Department of Medicine, DLuLswn of Oncology, Seattle, Washington 98195; the Fred Hutchinson Cancer Research Center, Seattle, Washington 98104; and the Sidney Farhw Cnncrr Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 Accepted
Ma-v
9, 1978
MSB-1 cells are a line of transformed chicken lymphoid cells derived from tumors induced by Marek’s disease viruses and free of exogenous avian leukosis viruses (ALV). They can be infected by ALV of subgroups A and C including transformation-defective (td) deletion mutants of avian sarcoma viruses (ASV). In terms of virus titers in supcrnatant culture medium, proportion of virus-producing cells, and levels of viral RNA detected by hybridization with a cDNA probe, infection by td ASV of MSB-1 cells was indistinguishable from infection of chicken embryo fibroblasts. In contrast, wild type ASV was restricted in its growth on MSB-1 cells. Different clones of ASV varied in their restriction by all these parameters of viral growth by factors of 10 ’ to 10 4. St,udies of a severely restricted viral clone showed equal quantities of hybridizable viral DNA in Hirt supernatant fractions of both fibroblasts and MSB-1 cells at 10 hr after high multiplicity infection, and transfection assays indicated infectious viral DNA in h&h cell types. Viral DNA largely disappeared from Hirt supernatant fractions of MSB-1 cells by 48 hr after infection, and sarcoma virusspecific DNA was not detected in Hirt pellet fractions from MSB-1 cells at levels found in comparably infected fihroblasts. Infectious ASV DNA, while easily detected in fihroblasts, could not be detected on MSB-1 cells at 48 hr or later times after infection. Because replication of td ASV does not appear restricted in MSB-1 cells, the failure of ASV DNA to integrate normally in these cells seems to be related to the presence of src sequences in the viral genome. INTRODUCTION
Our present understanding of the replication of exogenous avian leukosis-sarcoma viruses is based, for the most part, on studies employing cultured chicken embryo fibroblasts (CEF). As recently reviewed (Vogt, 1977), such studies have revealed three viral genes in both leukosis and sarcoma viruses involved in replication, gag (internal structural proteins), pal (reverse transcriptase), and env (envelope glycoproteins). These are the only known gene functions in field leukosis viruses, helper viruses found in stocks of defective sarcoma viruses, and transformation-defective (td) deletion mutants of nondefective sarcoma viruses. In contrast, the nondefective sarcoma viruses contain a fourth gene src which confers the ability to transform CEF 0042.SS22/78/08~-0~60$02.00/0 Copyright All rights
0 1978 hy Academic Press, of reproductwn m any form
Inc. reserved.
and a limited number of other cell types. Studies of both conditional and nonconditional mutants have not suggested any effect of src on viral replication in CEF. Analysis of the biology of these viruses in other cell types has begun recently. A line of transformed lymphoblasts called MSB-1 derived from a Marek’s disease virus (MDV) -induced lymphoma ( Akiyama and Kato, 1974) is suitable for such studies. These cells contain the MDV genome (Nazerian and Lee, 1974), although only l-2% of the cells contain virus and form MDV antigens (Akiyama and Kato, 1974). They also seem devoid of expression of avian leukosis virus (ALV) information, being negative in chf and gs antigen tests for viral envelope and internal structural proteins, respectively, and lack evidence of exoge-
RESTRICTION
nous ALV DNA sequences in hybridization assays (Akiyama and Kato, 1974; Nazerian et al., submitted for publication). They can, however, be exogenously infected by subgroups A and C ALV. In this study, we examined the properties of viral replication on MSB-1 cells and observed that the replication of avian sarcoma viruses (ASV) of subgroups A and C was markedly restricted in these cells while their corresponding td deletion mutants replicated normally. Further study suggested that, following infection of MSB-1 cells with ASV, infectious proviral sequences were synthesized, but the viral DNA did not integrate in host cell DNA in a normal fashion and was lost from the cell. Inasmuch as these abberations did not affect the replication of td ASV, they seemed to be due specifically to the presence of src. MATERIALS
AND
METHODS
Cells. CEF were cultured from chf embryos of the C/E and C/BE phenotypes obtained from H & N Farms, Redmond, Wash,, according to previously described techniques (Rubin, 1960). Transfection experiments utilized CEF of the same phenotype obtained from SPAFAS, Norwich, Conn. MSB-1 cells were contributed by Dr. Kevin Nazerian of the USDA Regional Poultry Research Station, East Lansing, Mich. They were cultivated in a COa incubator at 37” in RPM1 medium 1640 containing 10% fetal calf serum with 55 pg/ml penicillin, 35 pg/ml streptomycin, and 1 pg/ml amphotericin B (complete RPMI). These cells were seeded at 0.5 x lO’/ml and passaged by centrifugation and resuspension in fresh medium every 2 days when the cell density reached about 4 x lO”/ml. MSB-1 cells also formed colonies in soft agar with an efficiency of up to 50% when the following protocol was used. Hard overlay agar used in focus assays (see below) without dimethyl sulfoxide was used as a basal layer in 60-mm plastic tissue culture plates. About 100 MSB-1 cells per plate were overlaid in 3 ml of a medium containing 0.34% agar and 45% (by volume) complete RPMI, 4% tryptose phosphate broth, 10% fetal calf serum, 40% Ham’s medium F10, 1% 100X vitamin concentrate (GIBCO)
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ASV
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supplemented with (by weight) 0.1% sodium bicarbonate and 0.01% folic acid, Cultures were reoverlayed once at 6 days with 0.45% agar containing (by volume) 50% medium F-10 with 5% fetal calf serum and 50% complete RPMI. Viruses, ASV selected for study were Prague strain of Rous sarcoma virus of subgroups A and C (PR-A, PR-C) and the subgroup C virus, B-77. All sarcoma viruses were cloned from either foci or agar colonies. Among the PR-A viruses, clones lb, lc, 5, and 6 were produced by cells grown from individual foci of transformation while clones 2a, 4c, 5b, and 6b represented viruses produced by cells from agar colonies of CEF transformed by stock PR-A. Similarly, B77 clone 1 and PR-C clones 4a and lc were derived from foci, whereas PR-C, 4a clones 3, 11, 14, and 20 were all subclones of PR-C clone 4a derived from agar colonies. Leukosis viruses studied were td RSV, laboratory stocks of td PR-C and td PR-A, and newly isolated td PR-C lc recovered by endpoint dilution of parental PR-C clone lc grown on MSB-1 cells (as described in Resuits). Virus infection and assays. About 1 x 10” MSB-1 cells were suspended in 0.5 ml F-10 growth medium and infected with the appropriate dilution of virus for 20 min at 37”. For subgroup C viruses, 2 pg/ml polybrene (Aldrich) was present. The cells were then centrifuged, resuspended in RPM1 growth medium and cultured as described above. Supernatant culture medium derived from MSB-1 or CEF was assayed by focus assays for ASV and by the endpoint interference method for td RSV as previously described (Wyke and Linial, 1973; Linial and Neiman, 1974). For measuring the capacity of infected MSB-1 cells to form infectious centers, the following protocol was used. An appropriate number of infected cells were suspended in F-10 growth medium at a concentration of l-5 x lo”/ml and irradiated with 840 R from a “‘7Cs source. At this dose, all MSB-1 cells were killed. The irradiated cell suspension was then serially diluted and plated on freshly prepared monolayers of CEF. For ASV-infected MSB-1 cells, these monolayers were then overlayed 24 hr later and examined
362
NEIMAN
for foci of transformation in the standard fashion. The proportion of infective centers among the MSB-1 cells was estimated from the dilution and the number of foci counted. For td RSV-infected MSB-1 cells, plates of CEF innoculated with irradiated MSB-1 cells were passaged three times to ensure complete infection and then challenged with ASV of the same subgroup. The proportion of infective centers among the MSB-1 cells was then calculated from the dilution of MSB-1 cells at which interference was no longer detected. Assays of reverse transcriptase in culture supernatants. Concentration of virus particles released in cultures of infected MSB-1 cells and CEF was also estimated by measurement of reverse transcriptase levels in supernatant medium. Aliquots, 3-10 ml, of tissue culture fluid were clarified at 10,000 g for 10 min and virus sedimented from supernatant at 150,000 g for 30 min. Viral pellets were dissolved in reaction mixtures containing 50 mM Tris, pH 8.3, 1% NP-40, 10 mM dithiothreitol, 6 mM Mg(C&02)2, 60 mM NaCl, and 0.02 mM each of d ATP, d CTP, d GTP, and [“HITTP (50 Ci/mM). Reactions were stopped by the addition of lo%> trichloroacetic acid (TCA) and DNA synthesis assayed as cpm incorporated into TCA-precipitable material. The rate of reaction was assessed as PM of TTP incorporated/hr/ml of tissue culture fluid from the linear portion of the reaction curve (generally, 15 and 30-min samples showing a I:2 ratio of incorporated cpm). Control reactions for background determinations contained “viral pellets” from uninfected tissue culture fluid. Preparation and labeling of viral RNA and cDNA. Viral 70 S complex RNA was isolated from PR-C and labeled to a specific activity of 2-4 X 1Oi cpm as previously described (Neiman et al., 1974). The preparation and characterization of ‘“‘I-labeled fragments of the RSV genome composed of the 2000 nucleotides at the 3’-terminus is described elsewhere (Neiman et al., 1977). Single-stranded cDNA complementary to PR-RSV RNA was prepared as follows. PR-C 35s RNA, 1 pg [prepared as described previously (Neiman et al., 1977, 1978)], was incubated for 4 hr at 37” in a 50-~1 reaction
ET
AL.
mixture of 0.5 M Tris, pH 8.3, 0.18 M NaCl, 0.6 mu Mg(C2H302)2, 10 mM dithiothreitol, 0.125 pg/ml actinomycin D, 0.5 mM each of dATP, dCTP, and dGTP, 0.1 mM [“H]TTP (50 Ci/mmol) with 100 pg of DNase I limit digest calf thymus DNA fragments as primer (Taylor et al., 1976) and 20 units of AMV reverse transcriptase (a gift of Dr. Joseph Beard, 1 unit of enzyme incorporates 100 pM of TTP in 15 min). Reaction mixtures containing the digested DNA primer, but lacking template RNA, incorporated 0.002% of the quantity of acid-precipitable [“HIDNA product obtained in complete reaction mixtures. Reaction mixtures were diluted in 0.25 ml of 1 M NaCl, 0.1 M Tris, 0.01 M EDTA, pH 7.2, and extracted with an equal volume of chloroform-phenol mixture (l:l, v/v). The aqueous phase was diluted to 2 ml in 0.1 N NaOH and incubated in the dark at 20” for 16 hr. The solution was then neutralized and the DNA precipitated with 2 volumes of ethanol at -20”. The cDNA was dissolved in 20 ~1 5X SSC, 50% formamide containing 1 pg of RSV RNA and incubated for 24 hr. The reaction mixture was diluted in 5 ml of a solution of CsSOd in 2 mM EDTA with a density of 1.54 g/liter, centrifuged at 180,000 g for 60 hr, the gradient fractions in the region of hybrid density (1.48-1.62 g/liter) pooled and dialyzed against 0.01X SSC, the RNA hydrolyzed by treatment with 0.1 N NaOH, and finally the single-stranded cDNA product precipitated with 2 volumes of ethanol. The [“H]cDNA probe prepared in this fashion had a specific activity of about 2 X lo7 cpm/pg and an S1 nuclease sensitivity of 94%. When reacted with ““I-labeled RSV RNA in 5X SSC, 50% formamide at 50” for 24 hr (Cot value for cDNA = 2.7 X 10’) at cDNA:[““I]RNA mass ratios of 2:l and l:l, 22%) and 16%, respectively, of the viral RNA became resistant to pancreatic RNase. Conversely, about 75% of the cDNA probe was rendered resistant to S1 nuclease by hybridization with an excess of viral RNA. Preparation of cellular DNA. Fibroblasts or MSB-1 cells (2 X lO’/ml) were suspended in 0.1 M Tris, 0.001 M EDTA (T.E.) and lysed at 37” in the presence of 1% SDS. DNA was fractionated by the
RESTRICTION
method of Hirt (1967) by making the mixture 1 M NaCl and holding at 4” for 8-16 hr. The pellet fraction (“Hirt pellet”), obtained by centrifugation at 17,000 g for 15 min, was redissolved in T.E., and the DNA extracted with multiple spoolings on a glass rod by the method of Marmur (1971). The supernatant fraction (“Hirt supernatant”) was extracted with an equal volume of phenol/chloroform l:l, v/v) and precipitated with 2 volumes of cold ethanol. The pellet was redissolved in T.E. containing 0.1 M NaCl (standard buffer) and digested with 50 pg/ml pancreatic ribonuclease for 30 min at 37” followed by 50 pg/ml of pronase for 2 hr. The solution was then reextracted with an equal volume of phenol/chloroform and DNA precipitated from the aqueous phase with 2 volumes of cold ethanol. Preparation of cellular RNA. Recovery of RNA from MSB-1 cells was very poor in our hands using standard phenol-SDS or protease methods. Reasonable recoveries were obtained using the recently described guanidine extraction method (Strohman et al., 1977; Adams et al., 1977). MSB-1 cells (1 X 10” cells/ml) were suspended in 8 M guanidine-HCl, 10 mM Na(C2H302), pH 5.2, 1 mM dithiothreitol in a Dounce homogenizer and homogenized with 10 strokes of the “B” pestle. One-half volume of ethanol was added, the mixture held at -20’ for 30 min, and the RNA-containing material pelleted at 10,000 g for 10 min. The pellet was redissolved in 8 M guanidine-HCl, 10 mM Na(CnH:rOs), 20 mM EDTA, pH 5.2 (0.25 volume of the original homogenate), and reprecipitated with 0.5 volume of cold ethanol. This procedure was repeated three times. The final pellet was then dissolved in 20 mM EDTA, pH 7.0, and extracted with equal volumes of a 4:l chloroform/butanol mixture. The interface and organic phase were re-extracted in 0.5 volume of the EDTA solution and the two aqueous phases combined. This solution was made 3 M in Na(C&O.J, pH 5.2, and the RNA precipitated overnight in -20”. The RNA pellet was finally redissolved in 0.1 M Na( C,H,,O,), pH 5.2, and reprecipitated with ethanol. Recoveries were on the order of l-2 mg of RNA/5 X 10” MSB-1 cells which was dissolved in water and stored in liquid nitrogen
OF
363
ASV
until use. Hybridization conditions. All hybridization reactions with either [3H]cDNA or [1251]RNA probes were carried out in 0.75 M NaCl, 0.75 M C6H5Na307, 50% formamide solution at 50”, and in all cases, viral sequences in cellular DNA or RNA were present in excess. Hybrids were assayed as previously described by resistance to S1 nuclease when [“H]cDNA was used as a probe (Leong et al., 1972) and by pancreatic RNase when [‘““IIRNA was used as a probe (Neiman et al., 1974). The extent of reaction was measured as a function of CRot or Cot (mol-set/liter) based on either viral RNA or bulk cellular RNA or DNA concentrations when viral RNA or cellular RNA or Hirt pellet DNA was used as “driver.” When Hirt supernatant DNA was used as driver, hybridization was plotted as a function of C&t based on the number of cell equivalents present in the reaction mixture. The number of cells used for extraction was divided by the volume of the final DNA solution to obtain the number of cells represented per unit volume (cell equivalent, CE). The number of CE/pl in each reaction mixture was multiplied by the reaction time in seconds to obtain a value of CEot for each time point in the reaction. Individual reaction mixtures had concentrations of 1-3 x 10” CE/pl. Transfection assays. DNA from RSV-infected fibroblasts and MSB-1 cells were assayed for infectivity using the calcium method of Graham and Van der Eb (1973) as previously described (Cooper and Temin, 1976). Recipient cultures of CEF were exposed to 5 pg of total cell or Hirt pellet DNA preparations, or to approximately 1 X 10’ cell equivalents of Hirt supernatant DNAs in the presence of 5 pg of salmon sperm DNA. Foci of transformed cells were scored on the original DNAtreated plates. In addition, all recipient. cultures were tested for production of RSV either by transferring the cells or by assaying a sample of the supernatant medium on fresh CEF. RESULTS
Growth of ASV and td ASV on MSB-1 cells. MSB-1 cells were plated in soft agar
364
NEIMAN
and six clones were picked and established in culture, ASV and td ASV were used to infect these cells at a multiplicity of infection (m.o.i.) [based on focus-forming units (FFU) or interference units (IU) titered on CEF] of 0.03 and the supernatant culture fluids harvested at various times and assayed for FFU and/or IU on CEF and, in some cases, for reverse transcriptase levels. Figure 1 shows the growth of two agar derived clones of PR-A on six different clones of MSB-1 cells. Between 3 and 7 days after infection virus titers reach maximum levels which clustered for all MSB-1 cell clones around 10” FFU/ml (range, 104-10”) following infection with PR-A clone 2A and around 10” FFU/ml following infection with PR-A clone 6B. An exception was the somewhat higher titer (about lo4 FFU/ml) seen on clone IV MSB-1 cells. However, given the relatively uniform growth of a given ASV on the different cell clones, a single one, MSB-1 clone II, was selected for further analysis. Table 1 summarizes the titers of a panel of different ASV of subgroups A and C grown on MSB-1 clone II cells. A wide range of titers was observed with different virus clones ranging from very low levels to those approaching titers achieved on CEF. Where released virus particles were mea-
FIG. 1. Replication of RSV on different clones of MSB-I cells. MSB-1 cell stock was plated in soft agar as described in Materials and Methods and six clones; I, 0; II, q III, a; IV, A; V, 0; VI, n were picked, cultured, and infected with either PR-A clone 2a (panel A) or PR-A clone 6b (panel B) at an m.o.i. of 0.03. The infected cells were cultured in 2 ml of medium in X-mm plastic tissue culture dishes and the medium changed at 2.day intervals. The titer of FFU/ml in the supernatant medium was assayed and normalized for the number of cells in the culture at the time of assay.
ET
AL
sured by reverse transcriptase levels, virus production from MSB-1 cells seemed also reduced by a factor of lo”-lo4 as compared to that achieved on fibroblasts although the stoichiometry between enzyme levels and biological particles was, for unclear reasons, not precise. In contrast, virus titers from td RSV-infected MSB- 1 cells were uniformly near those achieved on fibroblasts. Thus, there appeared to be a restriction to replication of ASV on MSB-1 cells which varied from virus clone to virus clone and was either less apparent or did not apply at all to td deletion mutants of ASV. One consequence of this observation was that it was sometimes easy to recover td RSV from stocks of focus-cloned RSV simply by growth on MSB-1 cells presumably because low levels of such mutants in the sarcoma virus stock could replicate to high titer while the parental RSV remained restricted. For example, td PR-C lc was recovered by dilution of supernatant medium following infection of MSB-1 cells with the parental PR-C clone lc stock. As shown in Table 1, this virus replicated to about the same levels as stock td RSV in both fibroblasts and MSB-1 cells. Attempts to recover td RSV from freshly agar-cloned RSV by simple endpoint dilution were not successful. Effect of virus multiplicity on replication in MSB-1 cells. In this experiment, PR-C clone lc and td PR-C lc were compared. MSB-1 clone II cells were infected with either virus at various multiplicities. Six days after infection, supernatant medium was harvested for measurement of virus titers and appropriate dilutions of irradiated cells were plated on CEF for measurement of infectious centers. Figure 2 shows that for td RSV, the proportion of cells scoring as infectious centers increased linearly with increasing m.o.i. until the majority of cells yielded virus at an m.o.i. of about 0.1 (Fig. 2B). The titer of IU in the supernatant medium increased in proportion to the fraction of infectious centers (Fig. 2A). For the sarcoma virus, the proportion of infectious centers also increased linearly with m.o.i., but remained reduced lo:‘- to 104-fold in comparison with td RSV infection. Focus-forming activity in the super-
RESTRICTION
OF
TABLE VIRAL
Titer”
Virus
_ .___ -.. ~-~--.--~ Focus clones, subgroup PR-A clone lb PR-A clone 1 c PR-A clone 5 PR-A clone 6
2 x 1oi 4 x lOA >2 x 10’ 5 x 10’
A
Focus
C
clones
subgroup
RSV(RAV-7).2 B-77 clone 1 PR-C clone 4a
Reverse transcrip ~...~~ tase
A
Agar clones, subgroup PR-A clone 2a PR-A clone 4c I’R-A clone 5b I’R-A clone 6b
RSV(RAV-71-l
GROWTH
4.5 6.1 7.5 1.4
1
ON MSB-
x x x x
10” 10’ 10’ IO’
1.3 1.5 1 1
x x x x
10’ IO4 IO’ IO’
I CELLS’< .I‘i(er”
Virus
~~~~--Agar reclones PIT-(’ PR-C-4a-3 I’R-C-4a-11 PR-C-4a14 I’K-C-4a-20 Leukosis
8 3 4 1
365
ASV
td td trl
0.11 0.12
All
clone
4d’
7 x IO’
0.67
4 x 10’ 1 x IO’ 2 x IO
2.44 03; 1.74
viruses
I’R-A PR-C I’R-C-lc
viruses
Reverse transcrip. t ---asra’
5 x 10” 1 x 10” H x 10’ groum
on fibrobln.stL
0.42
” All infections were of MSB-1 clone II cells at an m.o.i. of 0.01 to 0.1. Assays were 5-7 days after infection. ” Titer FFU/ml for ASV or IU/ml for td ASV [for RSV (RAV-7), FFlJ/ml were measured]. ’ L’HlTTP incorporation in pmot/hr/ml of medium. ” PR-C clone 4a-infected fibroblasts were plated in soft agar, colonies picked and cult,ured. and supernatant virus from four colonies reassayed on MSB-1 clone II cells. ” Value from I’R-C-lc-infected CEF cultures.
natant medium also varied in proportion to the fraction of infectious centers about lO:‘fold below the titer achieved with td RSV. These data suggested that the small amount of RSV produced following infection of MSB-1 cells comes from a limited proportion of cells rather than from low level production by the majority of cells. Further, the linear relationship with m.o.i. up to a limit suggested the titration of a small fraction of either virus or MSB-1 cells capable of productive infection. If there existed a small proportion of virus infectious for MSB-1 cells in the RSV stock (maximally, 1 in 10:’ CEF infectious particles) they should have been easily eliminated by recloning the virus stock. As shown in Table 1, however, recloning of this virus by the agar colony technique did not eliminate the low level of RSV production. lJnless this focus-forming activity represented variants rapidly accumulating in the virus stock after cloning, the most reasonable interpretation seemed to be that RSV was produced by a small proportion of sus-
ceptible cells (far smaller than occurred with td RSV infection) which could be increased, somewhat, by increasing m.o.i. This idea was tested further by an experiment in which MSB-1 clone II cells were plated in soft agar (to reduce viral spread) immediately after infection at an m.o.i. of 2 with PR-C clone 4a. As might be expected from the experiment shown in Fig. 2, none of 10 resulting presumptively infected MSB-1 clones produced virus by assay of FFU or reverse transcriptase (data not shown). Cells from these clones could reasonably be assumed to represent the consequences of nonproductive infection of MSB-1 cells by RSV and were used along with cells from infected and uninfected masscultures, for further biochemical analysis. Viral RNA levels in MSB-I cells. Viral RNA levels in MSB-1 clone II cells were assessedbefore and after viral infection by hybridization with a [“H]cDNA to PR-RSV RNA. The results of that analysis are shown in Fig. 3 in which the extent of
366
NEIMAN
I
FIG. 2. Effect of multiplicity of infection. MSB-1 clone II cells were infected with either PR-C clone lc, 0, or td PR-C clone IC, 0, at various m.o.i., and the cells cultured for 6 days. Panel A shows the titer of IU, 0, or FFU/ml, 0, in the supernatant medium, and panel B shows the proportion of infective centers for the two viruses as a function of the initial m.o.i.
hybridization of the probe is plotted as a function of CRot based on the concentration of total cellular RNA in the reaction mixture. Under the conditions of these hybridization assays, the CRotlp observed with PR-RSV template RNA was about 8 x lo-“, whereas that of RNA from td RSVinfected MSB-1 cells was about 2 X 10” suggesting that about 0.4% of RNA from td RSV-infected cells was complementary to the viral cDNA probe. The slightly lower extent of hybridization in comparison to that seen with template RNA may be attributable to the absence of src-related RNA sequences in the td RSV-infected cells. This level of viral RNA is comparable to that reported for infected CEF using similar methodologies (Leong et al., 1972; Coffin and Temin, 1972; Hayward, 1977). In this connection, the figure demonstrates that we detected with this cDNA more viral RNA in uninfected MSB-1 cells (which are functionally gs- chf) than has been ob-
ET AL served in uninfected CEF with the same level of endogenous viral antigen expression (Hayward and Hanafusa, 1973; Hayward, 1977). Nevertheless, this RNA concentration is still (at a CRotlp of about 4 x 103) lower than that observed in gs+ chf+ fibroblast cultures. To the point of these experiments, Fig. 2 demonstrates that RNA from MSB-1 clone II cells infected at an m.o.i. of 2 and then recloned immediately in soft agar (see preceding section) contained no more RNA detectable with our cDNA probe than did uninfected cells. Cultures of MSB-1 clone II cells which were infected with RSV at the same high m.o.i. and grown for a week in standard culture contained a level of viral RNA roughly proportional to the titer of FFU in the supernatant at the time of extraction. For example, Fig. 2 shows an analysis of such cells infected with PR-C clone 4a at a point when the FFU titer in the culture was about 2 x 104/ml. The CRotlp observed was about lo”, corresponding roughly to a 50- to loo-fold lower viral RNA concentration that observed in td RSV infected cells. Thus, within the limits of the viral sequence representation of the cDNA, and the sensitivity of the method, we detected no clear evidence of transcription of exogenous viral genes in
FIG. 3. Concentration of viral RNA in infected MSB-1 clone II cells. About 1000 cpm (0.05 ng) of the cDNA probe to PR-RSV RNA was incubated for various time periods with either 1 ng RSV RNA, q 1 fig total cell RNA from td PR-C-infected MSB-1 cells, 0; 25 lrg total cell RNA from PR-C-4a subclone 3infected MSB-1 cells, 0; 50 g total cell RNA from PR-C-4a-infected and recloned MSB-1 clone II cells (see text), a; or 50 /~g total cell RNA from uninfected MSB-1 cells, W, in 5 ~150% formamide, 5X SSC at SO”. Hybridization was plotted as a function of CK,t (molset/liter) where CR,, = concentration of RNA nucleotides in mol/liter and t is the time in seconds.
RESTRICTION
the vast majority of RSV-infected MSB-1 cells. Detection of viral DNA in Hirt supernatant fraction. Inasmuch as we could detect no clear evidence of either the formation and release of virus particles or the accumulation of viral RNA in the large majority of MSB-1 cells following infection with ASV, it seemed reasonable to examine the early events after virus infection for evidence suggesting the mechanism of restriction of sarcoma virus replication. DNA preparations from Hirt supernatant fractions extracted from MSB-1 clone II cells and CEF 10 hr following infection with PRC clone 4a at an m.o.i. of 2 were analyzed by hybridization with the RSV cDNA probe. The results are shown in Fig. 4. As described in Materials and Methods, the data are compared on the basis of the number of cell equivalents contributing to the DNA in each reaction mixture. On this basis, the cDNA probe detected an apparently nearly equal concentration of complementary viral DNA sequences in Hirt su-
log
CE,t
FIG. 4. Viral DNA sequences in Hirt supernatant DNA. DNA from a portion of the Hirt supernatant fraction representing either 5 X 10” MSB-I clone II cells, * or CEF, 0; 10 hr after infection, or 8 x 10’ MS&l clone II cells 48 hr after infection with PR-C clone 4a at an m.o.i. of 2 was incubated with 1000 cpm of the PR-RSV cDNA probe under the same conditions as for Fig. 3. Hybridization is plotted as a function of C&t where CE,, = cell equivalents represented by the Hirt supernatant extract/PI of hybridization reaction mixture and t = time in seconds.
OF
ASV
367
pernatant fractions of both CEF and MSB1 cells 10 hr following infection. In contrast, the Hirt supernatant fraction from MSB-1 cells 48 hr after infection with the sarcoma virus contained very little DNA detectable with the cDNA probe. Using a more complex ‘““I-labeled viral genomic RNA probe, virtually no sequences were detected above background in this fraction (data not shown). Other investigators have reported a high concentration of apparently unintegrated proviral sequences in infected CEF persisting up to 3 days after infection (Khoury and Hanafusa, 1976), but, in MSB1 cells, we thus observed that the initially synthesized proviral sequences have largely disappeared by 48 hr after infection. Detection of viral DNA in Hirt pellet fractions. DNA was extracted from Hirt pellet fractions of MSB-1 cells 2 days and 16 days after infection with PR-C clone 4a at an m.o.i. of 2. In the latter case, clone II cells were recloned in soft agar immediately after infection, and clones were picked and cultured for 16 days to obtain an adequate number of cells for extraction as described before. A Hirt pellet fraction was also prepared 18 days after infection at an m.o.i. of 10. Because of the limited sequence representation in the cDNA probe, and the presence of endogenous RSV-related sequences in the normal chicken genome, an ‘““I-labeled genomic RNA probe was used which clearly distinguished between the DNA of infected and uninfected cells with respect to viral sequences (Neiman, 1972; Neiman et al., 19’74). For purposes of comparison, DNA from PR-C-transformed CEF and uninfected chick embryo cells was also analyzed. The kinetics of hybridization reactions between the lZ51viral RNA probe and these various DNA preparations are shown in Fig. 5. None of the MSB-1 cell DNA preparations formed hybrids with viral RNA with a rate or extent comparable with that observed with DNA from infected CEF (previously estimated to contain 2-4 proviruses/haploid genome) (Neiman, 1972; Wright and Neiman, 1974; Neiman et a!., 1974). The MSB-1 cells infected at an m.o.i. of 2 seemed to have no more reactive DNA than the uninfected cells. More hybridization was detected with MSB-1 cell DNA
368
NEIMAN
I
IO’
I03
104
105
co+
FIG. 5. Viral DNA sequences in Hirt pellet DNA. Panel A shows the kinetics of hybridization of 2oOO cpm ‘?-labeled PR-RSV RNA with 200 pg of cellular DNA from I’R-C-transformed fibroblasts, 0; uninfected CEF, 0; MSB-I clone II cells infected with PRC clone 4a at an m.o.i. of 2 48 hr after infection, e 16 days after infection and recloning, A; and 18 days after infection with the same virus at an m.o.i. of 10, & in 20 ~1 of hybridization mixture under the same condtions as described in the preceding figures. Panel B shows hybridization of “‘I-labeled 3’ end fragments of PR-C subunit RNA representing the terminal 2000 nucleotides of the viral genome with the MSB-1 Hirt pellet DNA from the m.o.i. = 10 infection used in panel A, m and from MSB-I cells infected with I’R-C clone 4a at an m.o.i. of 0.3, v. Conditions were as for panel A.
after infection at an m.o.i. of 10. To determine whether this increased hybridization reflected exogenously introduced sarcoma virus DNA sequences, a special ‘““I-labeled PR-C probe was prepared composed of sequences comprising the 2000 nucleotides at the 3’ end of the viral genome. This segment includes about 1500 nucleotides of the src gene of the virus and a terminal region shared with td RSV (Wang et al., 1975; Coffin and Billeter, 1976; Junghans et al., 1977; Neiman et al., 1977). In the presence of an adequate concentration of unlabeled competitor RNA from td RSV, and under these conditions of hybridization, the maRSV sequence jority of this ‘““I-labeled
ET
AL.
forms hybrids with DNA from RSV-infected CEF, whereas very little forms hybrids with normal chicken DNA as previously reported in detail (Neiman et al., 1977). This fragment could be used to attempt to detect RSV-specific sequences in MSB-1 cell DNA. Figure 5B shows that DNA from the MSB-1 cells infected at an m.o.i. of 10 formed hybrids with this srcspecific probe, but very little reaction was detected in MSB-1 cells infected at a lower multiplicity (0.3). The possibility exists that these sequences found in Hirt pellet DNA might be nonspecifically trapped, unintegrated viral DNA. This explanation seems, however, extremely unlikely since we have detected very little or no viral DNA in Hirt supernatant fractions at the intervals after infection when these analyses were performed (see Fig. 4). For example, the 3’ end fragment used as a src-specific probe formed no hybrids at all with Hirt supernatant fraction DNA obtained from the MSB-1 cells infected at an m.o.i. of 10 (data not shown). Thus, some RSV-specific sequences detected probably do either integrate or at least “associate” with chromosomal DNA in a manner leading to precipitation by the Hirt procedure. Nevertheless, the process did not occur as extensively in most RSV-infected MSB-1 cells as occurs in RSV-infected CEF, and appeared to require an extremely high m.o.i. Finally, the data obtained in this analysis of both Hirt supernatant and pellet fractions did not indicate whether complete or otherwise “normal” proviruses are found in most MSB-1 cells following infection with RSV. Formation of infectious DNA after infection with RSV. The infectivity of DNA extracted from PR-C-infected MSB-1 cells was assayed by transfection to determine whether complete copies of RSV DNA were present (Table 2). Hirt supernatant DNAs extracted 10 hr after infection of both CEF and MSB-1 clone II cells with PR-C clone 4a (m.o.i. = 2) contained infectious RSV DNA, although the DNA of PR-C-infected MSB-1 cells appeared less infectious than the DNA of PR-C-infected CEF. PR-C-infected CEF also contained infectious RSV DNA 48 hr after infection and 14 days after infection. In contrast, no infectious RSV
RESTRICTION TABLE INFECTIVITY
OF RSV
FIBROBLASTS
Donor cells CEF
MSB-1
DNA
2
DNA AND
OF PR-C-INFECTED
MSB-I
Hirt supernatant, Total cell, 48 hai Total cell, 14 dai
CELLS~’
Fraction positive recipient cultures’
preparationh
10 hai
Hirt supernatant, 10 hai Hirt pellet, 48 hai Total cell, 48 hai Hirt pellet, 18 dai
6/16
W3 9/12 2/lfi O/5 o/9 O/8
” Recipient cultures of CEF were treated with 5 ag of total cell or Hirt pellet DNA preparations, or with approximately 1 x 1Oi cell equivalents of Hirt supernatant DNA. Transformation was scored on the DNAtreated plates and supernatant media harvested 7 days after DNA treatment was assayed on fresh CEF to test for production of RSV. * DNAs were extracted at the indicated times after infection (hai, hours after infection; dai, days after infection) of CEF or MSB-1 clone II cells with PR-C clone 4a at an m.o.i. of 2, except for the Hirt pellet DNA extracted 18 days after infection of MSB cells at an m.0.i. of 10. ’ Number of DNA-treated cultures that produced progeny RSV over the total number tested.
DNA was detected 48 hr after infection of MSB-1 cells with PR-C. In addition, no infectivity was detected in assays of the Hirt pellet DNA of MSB-1 cells infected with PR-C at an m.o.i. of 10, although this DNA contained PR-C-specific DNA sequences detected by hybridization (Fig. 5). These results, therefore, indicated that infectious RSV DNA was synthesized in PRC-infected MSB-1 cells, but was present only at early times after infection. DISCUSSION
The principal conclusion from this set of observations is that the replication of ASV in most MSB-1 cells is restricted in comparison to their replication on fibroblasts. The data suggest that in the majority of MSB-1 cells, infection with ASV fails to result in the production of virus particles although in a small fraction of infected cells there appears to be production of focus forming virus. The fraction of cells capable of productive infection varies with different
OF
ASV
369
clones of ASV, and can be increased somewhat by high multiplicities of infection. In contrast, td deletion mutants of RSV replicate, in terms of virus titers, infectious centers, and intracellular concentrations of viral RNA to roughly equivalent levels on both MSB-1 cells and CEF strongly suggesting that the presence of src sequences in the ASV genome is critical for the operation of the MSB-1 cell restriction. It should be pointed out that these conclusions apply to viruses of subgroups A and C. Neither leukosis nor sarcoma viruses of other subgroups (e.g., B, D, E) will replicate in MSB-1 cells, presumably on the basis of a surface barrier. Our studies of the event in the virus life cycle where ASV replication is interrupted indicated that the restriction occurs as a result of a block in events occurring early after infection. Hybridization and transfection analyses suggested that presumptively unintegrated viral DNA sequences accumulated in apparently normal quantities by 10 hr after infection, and that this viral DNA is infectious. By 48 hr after infection, most of this free DNA is no longer detected in Hirt supernatant fractions, and, in contrast to the situation in ASV-infected fibroblasts, infectious DNA can no longer be detected in total cell DNA. It thus appears that while normal ASV DNA is initially formed in MSB-1 cells, this viral DNA is lost from the cell. Some ASV-specific sequences do become either closely associated with or integrated into chromosomal DNA in a large fraction of MSB-1 cells after infection at very high m.o.i. We did not obtain evidence, however, that these sequences represented infectious proviral DNA. The restriction of ASV replication on MSB-1 cells bears some resemblance to the Fv-1 restriction of murine leukemia virus replication, which appears to act before integration of viral DNA, but after synthesis of virus-specific DNA detectable by nucleic acid hybridization (Jolicoeur and Baltimore, 1976; Sveda and Soeiro, 1976). A mechanism is needed to explain how MSB-1 cells discriminate between the newly formed provirus of ASV and that of td ASV. The presence of a nuclease in MSB-1 cells (perhaps analogous to bacte-
370
NEIMAN
rial restriction endonuclease) which recognizes specific src DNA sequences is an attractive hypothesis which is compatible with all of the observations we have made and which may be testable. We have not as yet obtained any direct evidence of specific degradation of ASV DNA. Whatever the specific factors are that obstruct replication of ASV, it is clear that they are far more evident in MSB-1 cells than in CEF. It is possible that the mechanism for restricting ASV is simply absent in CEF. However, considerable variation was seen in the efficiency of restriction of different clones of subgroup A and C ASV by MSB-1 cells (see Table 1). Because ASV have been selected for growth on CEF for many years, these agents may have changed their src sequences sufficiently to overcome restriction by these cells. That ASV provirus integration occurs more readily in CEF than this lymphoid cell line may indicate at least part of the reason for the apparent specificity of fibroblasts and related cell types for in vivo transformation by ASV. We hasten to add, however, that in addition to being lymphoid, MSB-1 cells differ from CEF in being both transformed and infected with MDV. Which of these factors is significant in determining the specific restriction of ASV in MSB-1 cells remains to be established. ACKNOWLEDGMENTS We thank Don Macdonnell and Sharon Okenquist for excellent technical assistance. This investigation was supported by Grants CA 20068, CA 15704, and CA 18689, awarded by the National Cancer Institute. Dr. Neiman is a Scholar of the Leukemia Society of America. REFERENCES S. L., SOBEL, M. E., HOWARD, B. H., OLDEN, K., YAMADA, K. M., DE CROMBRUGGHE, B., and PASTEN, I. (1977). Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Nut. Acad. Sci. USA 74, 3399-3403. AKIYAMA, Y., and KATO, S. (1974). Two cell lines from lymphomas of Marek’s disease. B&en J. 17, 105-116. COFFIN, J. M., and TEMIN, H. M. (1972). Hybridization of Rous sarcoma virus DNA polymerase product and ribonucleic acids from chicken and rat cells infected with Rous sarcoma virus. J. Viral. 9, ADAMS,
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766-775. COFFIN, J. M., and BILLETER, M. A. (1976). A physical map of the Rous sarcoma virus genome. J. Mol. Biol. 100, 293-318. COOPER, G. M., and TEMIN, H. M. (1976). Lack of infectivity of the endogenous avian leukosis virusrelated genes in the DNA of uninfected chicken cells. c1. Vmll. 17, 422-430. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,456-467. HAYWARD, W. S., and HANAFUSA, H. (1973). Detection of avian tumor virus RNA in uninfected chicken embryo cells. J. Viral. 11, 157-167. HAYWARD, W. S. (1977). Size and genetic content of viral RNAs in avian oncovirus-infected cells. J. Virol. 24,47-63. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369. JOLICOEUR, P., and BALTIMORE, D. (1976). Effect of Fv-I gene product on proviral DNA formation and integration in cells infected with murine leukemia viruses. Proc. Nat. Acad. Sci. USA 73, 2236-2240. JUNGHANS, R. P., Hu, S., KNIGHT, C. A., and DAVIDSON, N. (1976). Heteroduplex analysis of avian RNA tumor viruses. Proc. Nat. Acad. Sci. USA 74, 477-481. KHOURY, A. I., and HANAFUSA, H. (1976). Synthesis and integration of viral DNA at different times after infection with various multiplicities of avian oncoviruses. J. Virol. 18, 383-400. LEONG, J. A., GARAPIN, A., JACKSON, N., FANSHIER, L., LEVINSON, W. E., and BISHOP, J. M. (1972). Virus-specific ribonucleic acid in cells producing Rous sarcoma virus: Detection and characterization. J. Viral. 9, 891-902. LINIAL, M.. and NEIMAN, P. (1976). Infection of chick cells by subgroup E viruses. Virology 73, 508-520. MARMUR, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3, 208-223. NAZERIAN, K., and LEE, L. F. (1974). Deoxyribonucleic acid of Marek’s disease virus in a lymphoblastoid cell line from Marek’s disease tumour. J. Gen. Virol. 25, 317-321. NEIMAN, P. E. (1972). Rous sarcoma virus nucleotide sequences in cellular DNA: Measurement of RNADNA hybridization. Science 178, 750-753. NEIMAN, P. E., WRIGHT, S. E., MCMILLIN, C., and MACDONNELL, D. (1974). Nucleotide sequence relationships of avian RNA tumor viruses: Measurement of the deletion in a transformation-defective mutant of Rous sarcoma virus. J. Viral. 13,837-846. NEIMAN, P. E., DAS, S., MACDONNELL, D., and MCMILLIN-HELSEL, C. (1977). Organization of shared and unshared sequences in the genomes of chicken endogenous and sarcoma viruses. Cell 11, 321-329.
RESTRICTION
OF
371
ASV
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“Comprehensive Virology,” Vol. IX. pp. 34 I-455 (H. Fraenkel-Conrat and Ii. Wagner, eds.). Plenum Press, New York. WANG, L. H., DUESBERG, l’., BEEMON, K., and VOGT, 1~. K. (1975). Mapping T,-resistant oligonucleotides of avian tumor virus RNAs: Sarcoma specific oligonucleotides are near the poly(A) end and oligonucleotides common to sarcoma and transformation defective viruses are at the poly(A) end. ,J. Viral.
M. M., and SOEIRO, R. (1976) Host restriction of Friend leukemia virus: Synthesis and integration of the provirus. Proc. Nat. Acad. Sci. USA 73,
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H. (1960). A virus in chick embryos which induces resistance in uiko to infection with Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 46, 1105-1119. STROHMAN, R. C., Moss, P. S., MICOU-EASTWOOD, J., SPECTOR, D., PRZYBYLA, A., and PATERSON, B. (1977). Messenger RNA for myosin polypeptides: Isolation from single myogenic cell cultures. Cell 10, KUBIN,
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2356-2360. It., and SUMMERS, .J. (1976). Efficient transcription of RNA into DNA by avian sarcoma virus polymerase. Biochim. Biophys. AIfrr 442, 324-330. VOG~, P. K. (1977). Genetics of RNA tumor viruses. TAYLOR,
.J. M.,
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S. E., and relationships
NEIMAN,
between
P. E. (1974). Base-seavian ribonucleic acid
endogenous and sarcoma viruses assayed petitive ribonucleic acid-deoxyribonucleic bridization. Biochemistry 13, 1549-1554. WYKE, tive
J., and
LINIAL,
avian sarcoma terization of twenty
by comacid hy-
M. (1973). Temperature-sensiviruses: A physiological characmutants. Virology 53, 152-361.