VIROLOGY
93, 20-30
(1979)
Transformation by Reticuloendotheliosis Virus: Development of a Focus Assay and Isolation of a Nontransforming Virus JACALYN
D. HOELZER,’
Department
RAY B. FRANKLIN,
of Microbiology,
The University
AND
of Texas at Austin,
HENRY R. BOSE, JR. Austin,
Texas 7871i?
Accepted October 6, 1978
A focus assay for quantitating in vitro transformation by oncogenic reticuloendotheliosis virus (REV) has been developed in Japanese quail embryo fibroblast cultures. The titer of the transforming virus detected in the in vitro focus-forming assay correlates with the development of reticuloendotheliosis in chickens. The titration pattern of REV focus formation appears to follow two-hit kinetics. A nontransforming member has been identified in the REV stocks and is designated reticuloendotheliosis-associated virus (REV-A). The nontransforming virus, REV-A, is present in REV stocks in lOO-to lOOO-foldexcess over the transforming member. REV-A causes an initial cytopathic effect in chick and quail embryo fibroblast cultures. The surviving cells continue to divide leading to the development of a persistently infected culture. The persistently infected cultures are morphologically indistinguishable from uninfected avian fibroblast cultures. REV-A interferes with superinfection by the oncogenic member. The addition of REV-A to oncogenic preparations of REV increases focus formation and changes the titration pattern from two-hit to one-hit kinetics indicating that REV is defective.
occurs after a brief latent period (Temin and Kassner, 1974). Following this acute Members of the reticuloendotheliosis (RE) phase the cytopathic effect disappears and group of retroviruses have been isolated the cultures chronically yield virus (Temin from a number of avian species. These and Kassner, 1975). These persistently inviruses have similar morphology (Kang fected cultures are morphologically similar et al., 19’75),RNA and polypeptide composi- to uninfected fibroblasts. tion (Maldonado and Bose, 19751,a groupReticuloendotheliosis virus (REV), the reactive antigen (Maldonado and Bose, 19’761, prototype, is the only member presently and they share extensive RNA sequence included in the RE group shown to be capable homology (Kang and Temin, 1973). At the of oncogenic transformation. REV induces present time, members of the RE group can an acute systemic proliferative disease with be distinguished only on the basis of patho- distinctive lesions composed of reticulogenicity and cross-neutralization tests which endothelial cells in the liver, spleen, and reflect differences in virion surface glycopro- other visceral organs of experimentally teins (Purchase and Witter, 1975). These infected chickens (Sevoian et al., 1964; viruses induce a variety of syndromes Thielen et aZ., 1977; Olson, 1967). REVincluding spleen necrosis, anemia, visceral transformed cell lines have been isolated, reticuloendotheliosis, and lymphoid nerve cloned, and cultured from the bone marrow lesions (Purchase et al., 1973). When chick of REV-infected birds (Franklin et al., 1974). embryo fibroblast (CEF) cultures are in- The virus produced from these bone marrow fected with RE viruses, virus production cultures (BMC) transforms CEF cultures with the development of a cytopathic effect in vitro (Franklin et al., 1977). RNA tumor viruses are divided into ’ To whom reprint requests should be addressed. sarcoma and leukemia viruses based on their INTRODUCTION
0042-6822/79/030020-l1$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
20
REPLICATION-DEFECTIVE
oncogenic potential (Vogt, 1965). The leukemia viruses generally do not morphologically transform cells in culture. However, several viruses in addition to REV have been described which induce acute lymphoproliferative diseases and are capable of transforming cells in vitro. Avian myeloblastosis virus transforms bone marrow and yolk sac cultures (Beaudreau et al., 1960; Baluda and Goetz, 1961; Moscovici, 1967; Moscovici et al., 1975) and avian erythroblastosis virus strain R (AEV-R) (Ishizaski and Shimizu, 1970) and myelocytomatosis virus (MC29) transform CEF cultures (Langlois and Beard, 1967). Abelson mouse leukemia virus also transforms both bone marrow and mouse fibroblasts (Rosenberg et al., 1975; Scher and Siegler, 1975). These in vitro transforming leukemia viruses are replication defective (MC29, Ishizaki et al., 1971; AMV, Moscovici, 1975; AEV, Graf et al., 1976; Abelson, Scher and Siegler, 1975). In this paper we report the development of a transformation assay for reticuloendotheliosis virus. This assay permitted us to demonstrate that oncogenic stocks of REV are composed of a mixture of transforming and nontransforming viruses. MATERIALS
AND METHODS
Viruses. Unipore (Bio-Rad) filtered (0.4 pm) supernatant fluids from cultures of the REV-transformed chicken bone marrow cell line (BMC) harvested in log phase was the source of oncogenic REV used in these experiments. These REV preparations were free of particles containing avian leukosis gene sequences (Franklin et al., 1977). The plaque assay of Moscovici and co-workers (1976) has shown that this fluid contains 5 x 10” plaque-forming units (PFU)/ml. Assay in vivo revealed 1 x lo4 LD,,jml for l-day-old Hyline chickens. The nontransforming virus present in the oncogenic REV stock was prepared by infection of secondary SPAFAS chicken or Japanese quail embryo fibroblast cultures with a lop5 dilution of the oncogenic REV stock. The cells were subcultured once and culture fluid was harvested at 5 days after infection.
TRANSFORMING
REV
21
Cell cultures. The REV-transformed BMC line was maintained in RPM1 1640 medium supplemented with 8% heat-inactivated fetal bovine serum. These BMC cultures are free of mycoplasma and Marek’s disease virus antigens (R. L. Witter, personal communication). Fibroblast cultures were grown in RPM1 1640 supplemented with 10% tryptose phosphate broth and 5% heat-inactivated calf serum. Focus assay for REV on quail embryo jibroblast cultures. Primary quail embryo
fibroblast (QEF) cultures were subcultured into 60-mm tissue culture dishes (Falcon) at a density of 8 x lo5 cells/plate in 5 ml of RPM1 1640 with 10% tryptose phosphate broth and 1% heat-inactivated calf serum. After 4 hr the medium was removed. The cultures were infected with dilutions of the oncogenic REV preparation in 1 ml of medium containing 25 pglml DEAE-dextran. Duplicate plates were infected for each dilution tested. After a 1-hr adsorption period at 37”, each plate was overlayed with 7 ml of a 0.7% agar mixture containing Ham’s FlO with 0.7% Bacto-Agar, 5% heat-inactivated calf serum, 1% heat-inactivated chick serum, 1% beef embryo extract, 0.028% sodium bicarbonate, 700 U penicillin1600 U streptomycin, and 0.01 mg gentamycin. Five days after infection, a second agar overlay (2 ml) containing the same components was added. Foci were scored on the eighth day after infection. Isolation in the REV
qf the
nontransforming virus stock by end point dilution.
Secondary QEF or CEF cultures were infected with several dilutions from 10-l through lo+’ of the oncogenic REV stock according to the method described above. After adsorption, the virus inoculum was removed and replaced with medium containing 10% tryptose phosphate broth and 5% calf serum. Three days after infection each plate was subcultured into duplicate 60-mm plastic petri dishes. The medium was changed on the fourth day and the virus-containing culture fluids were harvested on the sixth day. The virus present at each dilution was assayed for transforming ability in QEF, and in l-day-old chickens as described previously (Bose and Levine, 1967).The number
22
HOELZER,
FRANKLIN,
AND BOSE
RESULTS of virus particles in each dilution was also detected by the reverse transcriptase Development of a Transformation Assay assay. for Reticuloendotheliosis Virus The standard exogenous reaction (Waite and Allen, 1975) was carried out in a final volume of 0.1 ml containing 100 mM NaCI, The replication of RE group viruses in 50 mM Tris-HCI (pH 8.3), 5 mM dithio- CEF cultures results in an initial acute threitol (DTT), 0.05 mM manganous acetate, cytopathic effect (Temin and Kassner, 1974). 7.5 piIf poly(rA), 1.9 @l4 oligo(dT) 12-18, This property has allowed the development 0.2% Triton x-100, and 20 #.IJi of [3H]dTTP of a quantitative plaque assay for RE group ([3HJthymidine triphosphate; 40 to 60 Ci/ viruses (Temin and Kassner, 1974;Moscovici mmol). Virus in the culture fluid was pelleted et al., 1976). This assay does not measure in a SW41 rotor for 45 min at 30,000 rpm. the transforming potential of the protoThe virus pellet was suspended in 0.2 ml type REV. buffer and lo-,ul virus samples were mixed REV transforms avian bone marrow and with the reaction mix and incubated at 3’7 spleen cells in vivo (Franklin 1974;Kang and for 1 hr. At the end of incubation 2 ml of Temin, 1974)and in vitro (Hoelzer and Bose, cold TP (0.4 M trichloracetic acid, 0.02 M unpublished). A continuous line of REVsodium pyrophosphate) was added to each transformed chicken bone marrow cells has tube. The precipitate was collected on been maintained for over 4 years in this 0.45~pm Millipore filters, washed, dried, laboratory. This line chronically releases and counted in Bray’s scintillation fluid. oncogenic REV which transforms CEF culVirus interference assay. A stock of the turesin vitro (Franklin et al., 1977).Efforts to nontransforming member of REV was used quantitate foci in CEF cultures have met to infect secondary QEF cultures. Stocks of with only limited success. Since MC29 prothe nontransforming virus were diluted duces much more distinct foci on QEF culfivefold since this virus induces an extensive tures than on CEF cultures (Bister et al., cytopathic effect in fibroblast cultures. The 1977), QEF cultures were tested to deterplates were subcultured on the third and mine whether a quantitative transformation sixth days after infection and then infected assay could be developed for REV. Subconwith an oncogenic REV preparation at a fluent secondary QEF cultures were infected multiplicity of appoximately five. Secondary with serial dilutions of REV. Small discrete uninfected QEF cultures were also infected foci of transformed cells appeared on QEF at this time with the oncogenic REV stock. cultures 8- 10 days after infection. The REV The medium was changed on the eighth day foci were composed of disoriented, densely and virus-containing culture fluids were packed, spindle-shaped cells rather than the harvested 10 days after infection. The trans- rounded cells observed in a Rous sarcoma forming virus in these cultures was quanti- virus focus. Fig. 1 is a photograph of the tated by the focus-forming assay described cellular morphology of a REV-transformed above. focus in a confluent monolayer culture Kinetics offocus formation of REV in the of QEF. presence of excess nontransforming virus. The titration of an oncogenic REV stock A series of dilutions of the oncogenic REV obtained from the BMC line is shown in stock obtained from the BMC line was made Fig. 2. At higher dilutions the number of and distributed to duplicate sets. A twofold foci falls proportionally with the square of dilution of the nontransforming virus was the dilution factor indicating two-hit kinetics added to one set in a ratio of 1:l. Medium (Hartely and Rowe, 1966). Numerous titrawas added to each dilution of the second set tions of culture fluid obtained from different in place of the nontransforming virus. These BMC clones ranged from lo3 to lo4 focussets of inocula were then used to infect QEF forming units (FFU)/ml. Titration of REV cultures for use in the REV focus-forming produced from several BMC clones in Hyline assay. chickens give an average LD,, of approxi-
REPLICATION-DEFECTIVE
FIG. 1. An REV focus formed on a confluent monolayer mixture 8 days after infection.
mately 104LD,dml. Therefore, there exists a correlation between the development of reticuloendotheliosis in birds and the transforming activity measured in this in vitro focus-forming assay.
TRANSFORMING
REV
of quail embryo fibroblasts
23
overlayed
with 0.74 ) agar
Isolation of a Nontransforming Oncogenic REV Stocks
Viru .s in
As shown in Fig. 2, the transform ning activity of REV in QEF cultures appea rs to
HOELZER,
24
FRANKLIN.
AND BOSE
REV preparations contain a mixture of transforming and nontransforming viruses. Included in the RE group are a number of viruses which fail to transform fibroblast cultures or induce neoplastic diseases in experimentally infected animals. In this group are Trager duck spleen necrosis virus, chick syncytial virus, and duck infectious anemia virus. At the present time we are not certain whether the nontransforming virus present in the oncogenic REV stocks is one of these previously identified agents. With this reservation, we will refer to the nontransforming virus as reticuloendotheliosisassociated virus (REV-A). . It
10-27.5
50
2 5 IO-37550 CONCENTRATION REV STOCK
I
I
25 OF
Characterization of the Nontransforming Virus in the Oncogenic REV Stock
IO-4
FIG. 2. The dose-response relationship between the concentration of REV and the number of transformed foci formed on QEF cultures. Each point represents the number of foci formed by infection with 1 ml of the oncogenic REV stock obtained from the BMC line at the indicated dilutions. These results were obtained from the average of duplicate cultures at 8 days after infection.
follow two-hit kinetics at higher dilutions. This suggests that the oncogenic stocks obtained from the BMC line contains a mixture of two viruses (Hartely and Rowe, 1966). To determine whether the oncogenic REV stock contains a mixture of two viruses, lo-fold dilutions were used to infect secondary CEF and QEF cultures. The infected plates were subcultured on the third day and the virus-containing culture fluids were harvested on the sixth day. The presence of virus in the culture fluid was detected by a reverse transcriptase assay. The results are shown in Table 1. Infectious virus was detected up to a lop7 dilution. This number correlates with the results obtained by Moscovici and co-workers (1976) of 5 x lo6 PFU/ml of BMC REV stock. The transforming activity of this stock has an average of 5 x lo4 FFU/ml. Since there are approximately lo7 infectious virus particles in the stock these results indicate that oncogenic
When the nontransforming virus (REV-A) is used to infect QEF cultures as extensive cytopathic effect develops. By the fifth day after infection over 80% of the cells have lysed. Those cells which survive this acute phase of infection recover, and this leads to the establishment of a persistently infected culture. These results are shown in Fig. 3. The persistently infected cultures are morphologically indistinguishable from uninfected quail embryo fibroblasts. As shown TABLE
1
REVERSE TRANSCRIPTASE ACTIVITY OF CEF CULTURES INFECTED WITH DILUTIONS OF AN ONCOGENIC REV STOCK
Dilution” 100 10-S 10-c 10-7 10-s Background
[SH]TdR triphosphate incorporation* 11,383 14,983 1,968 941 291 187
fl Dilution of oncogenic REV used to infect chicken embryo fibroblasts (1 ml/GO-mm plate). b Counts per minute of [3H]thymidine triphosphate in a lo-p1 sample obtained from a detergent-disrupted virus pellet from 10 ml of CEF culture fluid 5 days after infection.
REPLICATION-DEFECTIVE
TRANSFORMING
25
REV
The Nontransforming Member Interferes with Replication of the Oncogenic Virus
The ability of leukosis viruses to interfere with sarcoma viruses of the same subgroup is well documented and serves as a measure of genetic relatedness (Vogt, 1965). To determine whether the nontransforming virus in the oncogenic REV stock interferes with the replication of the transforming virus, QEF cultures were infected with REV-A. The infected cells were subcultured 3 days after infection and challenged with oncogenic REV 6 days after infection. An uninfected set of secondary QEF cultures was also infected with the oncogenic REV stock at that time. Five days after superinfection with the oncogenic REV, the virus0
12345678 DAY
9 AFTER
IO
INFECTION
FIG. 3. The cytopathic effect induced by the nontransforming virus (REV-A) on QEF cultures. Freshly transferred secondary QEF cultures (6.5 x lo5 cells/ 60-mm petri dish) were infected with 1 ml of REV-A stock at a dilution of loo (0) or 10-l (0) in the presence of 25 @g/ml DEAE-dextran. Control plates received only 25 pg/ml DEAE-dextran. After a 1-hr adsorption period the inocula were removed and 5 ml of medium with 10% tryptose phosphate and 5% calf serum was added to each plate. The cell numbers were determined each day for duplicate plates. After trypsinization the cells were counted with the use of a hemocytometer. The medium was changed each day on the remaining cultures. The results are expressed as percentage control for each day.
in Fig. 4, the persistently infected cultures chronically yield virus as detected by the reverse transcriptase assay. The virus released from the persistently infected cultures fails to transform either CEF or QEF cultures. The response of avian fibroblasts to this nontransforming virus present in the oncogenic REV stock is similar to that described for other RE group viruses (Temin and Kassner, 1974). When REV-A is injected into l-day-old chickens they fail to develop the hepatosplenomegaly characteristic of REV. The latent period becomes protracted and the birds develop an acute runting disease accompanied by paralysis (data not shown).
:a5 0”I 6
4
0
12345678 DAY
9 AFTER
10
INFECTION
FIG. 4. The time course for replication of REV-A is avian fibroblast cultures. Cultures were infected with REV-A and virus production was measured by the release of particles with reverse transcriptase activity. Medium was harvested each day from duplicate cultures infected with an REV-A stock at a dilution of lOa (0) or 10-l (0). Virus-containing culture fluid (10 ml) was pelleted and suspended in 0.2 ml buffer. Samples (10 $1 were tested for reverse transcriptase activity as described under Materials and Methods. The results are expressed as counts per minute of [3H]tymidine triphosphate incorporated into acid-precipitable material from lo-p1 samples.
HOELZER, FRANKLIN,
26
AND BOSE
TABLE 2 ESTABLISHMENT OF INTERFERENCE BY REV-A AGAINST ONCOGENIC REV
Dilution
Cultures infected with REV”
Cultures infected with REV-A and superinfected with REV”
10” 10-1 10-Z 10-3 10-d IO-” 10-e
TNTC’ TbiTC TNTC 28,19 1,3 010 090
14,lO 030 030 0 090 030 030
Cultures infected with REV-A o,o 090
(1Culture fluid from QEF cultures taken on the fifth day after infection with oncogenic REV (1 ml/60-mm plate). h Culture fluid from QEF cultures 10 days after infection with the nontransforming virus and 5 days after superinfection with oncogenic REV. ’ Too numerous to count.
containing culture fluids were harvested and titrated for focus-forming ability in QEF cultures. The results are shown in Table 2. Stocks obtained from the BMC line or from the oncogenic stock passaged one time in QEF cultures had titers of approximately lo4 FFU/ml. Cultures that were previously infected with the nononcogenic member produced approximately 10 foci at the highest concentration or 0.1% as many foci. These results indicate that the nontransforming virus interferes with the replication of oncogenic REV. Change in the Kinetics of REV Focus Formation with Addition of Excess Nontransforming REV
Since the oncogenic REV stocks contain both a transforming and a nontransforming virus we tested the hypothesis that the transforming virus was defective. Duplicate sets of QEF cultures were infected with serial dilutions of an oncogenic REV preparation. A fixed amount of REV-A was added to each dilution of the oncogenic stock and used to infect one set of plates. The duplicate set of cultures was infected with serial dilutions of the oncogenic stock to which no REV-A was added. These cultures were overlaid with agar and foci were counted 8 days after infection. As shown in Fig. 5, addition of REV-A increases the number of
foci formed. In the presence of excess nontransforming virus, focus formation by REV is converted from two-hit to single-hit kinetics. The conversion from two-hit to single-hit kinetics suggests that REV is replication defective and that the transformed foci are formed by the infection of neighboring cells by REV. Consistent with the hypothesis that oncogenic REV is replication defective, we have isolated clones of REV-transformed non-virus producing QEF. Oncogenic REV can be rescued from non-virus producing transformed cells by infection of these cultures with the nononcogenic members of the RE group. DISCUSSION
The RE group of avian retroviruses consists of four members of which only one causes leukemogenic transformation. REV, the prototype of the group, is able to transform cells present in the bone marrow and spleen of chicken and quail (Franklin et al., 1974, Kang and Temin, 19’74). REV also transforms chicken and bone marrow cells in vitro and these cells are morphologically identical to those isolated and subsequently cultured directly from infected birds (Hoelzer and Bose, unpublished). These bone marrow cell lines are therefore presumed to represent the target cell for REV.
REPLICATION-DEFECTIVE
I
10475
5.0
2.!j IO375 50 CONCENTRATION REV STOCK
2.5 OF
IO-’
FIG. 5. The dose-response relationship of REV focus formation on QEF cultures in the absence (0) and presence (0) of added REV-A. Each point represents the number of foci formed by 1 ml of oncogenic REV stock at each dilution. These results were obtained from the average of duplicate cultures 8 days after infection. Corrections were made for change in volume of inocula due to addition of REV-A or media at each dilution.
The virus produced by the bone marrow cell line in vitro is oncogenic in birds (Franklin et al., 19’74)and transforms CEF cultures (Franklin et al., 1977). In order to understand the mechanism of transformation by this acute leukemogenic virus, a quantitative transformation assay has been developed. Distinct foci appear on QEF cultures. A correlation exists between the quantitation of REV in QEF cultures determined by this focus-forming assay and the development of reticuloendotheliosis in chickens. Focus formation in these QEF cultures appears to follow two-hit kinetics at higher dilutions. This suggests that the development of foci depends on infection of the cell by more than one virus particle. If infection by two viruses is necessary for successful focus formation the chance of the event occurring will be dependent on the product of the chance of each event occurring alone. Thus the number of foci will fall proportionally with the square of the virus dilution. The apparent two-hit kinetics observed with REV in the QEF
TRANSFORMING
REV
27
focus assay can be explained two ways. Either transformation requires the expression of two viral genomes or, alternatively, the virus with transforming activity is replication defective. The oncogenic stocks of REV contains a mixture of transforming and nontransforming viruses. When REV is titrated in the presence of excess nontransforming virus the dose-response curve is converted from a two-hit to a single-hit pattern. This suggests that the two-hit kinetics of focus formation represents the requirement of dual infection of a cell by both the oncogenic and noncogenic viruses in order to produce a visible focus. Therefore the oncogenic virus is presumed to be replication defective but contains the genetic information required for transformation. Serial passage of oncogenic REV in CEF cultures quickly leads to attenuation of the oncogenic disease potential (Halpern et al., 1973; Witter et al., 1970). The apparent attenuation of REV may simply reflect the loss of the transforming virus from the stock. Since REV transforms cells of hematopoietic origin the attenuation of REV during serial propagation of CEF cultures may result because the target cell for transformation is not present. This may account for the failure of other laboratories to detect transformation by REV in fibroblast cultures (Temin and Kassner, 1974; Witter et al., 1970). The nontransforming virus, designated REV-A, is present in the oncogenic stock in a lOO- to lOOO-foldexcess. Replication of REV-A in avian fibroblast cultures leads to an initial acute cytopathic effect. The plaque assays described for REV by other laboratories (Moscovici, 1976; Temin and Kassner, 1974) were measuring the effect of the nontransforming virus present in the REV stock. Similar cytopathic effect appears in avian fibroblast cultures infected with other members of the RE group and has permitted the development of a plaque assay for these viruses as well (Temin and Kassner, 1974). Those cells which survive the acute phase divide and form a chronically infected culture. The persistently infected cultures are indistinguishable from uninfected fibroblast cultures. REV-A fails to induce reticuloendotheliosis when injected in chickens. The infected birds
28
HOELZER, FRANKLIN,
develop an acute runting disease and after a protracted latent period death occurs (Hoelzer and Bose, unpublished). When oncogenic stocks of REV are inoculated into line N and P chickens, line N chickens develop reticuloendotheliosis. By contrast, line P chickens fail to develop this oncogenic disease (Scofieldet al., 19’78).Line P chickens appear to restrict tumor development by the oncogenic REV member. Line P chickens, however, develop an acute runting disease similar to that observed with REV-A. In addition to providing a function(s) to permit the production of infectious REV, REV-A may also play a significant role in the pathogenesis induced by REV. REV is an extremely virulent retrovirus. The incubation period of REV may be as short as 5 days and the mortality of experimentally infected animals approaches 100% (Sevoian et al., 1964). Recently, we have reported that the ability of both T and B (Scofield and Bose, 1978; Carpenter et al., 1977) cells to respond to mitogens becomes cytostatically suppressed during the first 3 days after infection of chickens with oncogenic preparations of REV. This suppression can be transferred to splenic lymphocytes obtained from uninfected chickens with spleen cells from infected birds by a contact-mediated event involving a trypsin-sensitive surface protein (Carpenter et al., 1977). The suppressed T cells obtained from REV-infected birds fail to undergo cell division but are fully capable of cytotoxic effector function (Carpenter et al., 1978). The suppressor cells do not express REV antigens (Carpenter et al., 1978). It seemsreasonable to postulate that the rapid generation of the contactmediated cytostatic suppressor contributes to the extreme virulence of REV. The early appearance of the suppressor should prevent maximal T cell-mediated antitumor responses by limiting lymphoid cell proliferation. Infection of birds with REV-A, which replicates in the chicken but does not induce reticuloendotheliosis, induces this suppressor cell population (Carpenter et al., unpublished). Thus REV-A may contribute to the extreme virulence of REV by suppressing the host cell-mediated response. Acute avian leukemia viruses induce neo-
AND BOSE
plasms in vivo at a high rate in a short period of time. They cause myeloid and erythroid leukemias and some can induce fibrosarcomas (Beard, 1963; Gross, 1970; Purchase, 1972; Hanafusa, 1977). These avian viruses that induce leukemia have the capacity to transform cells of hematopoietic origin and fibroblasts in. vitro (MC29, Graf, 1973;AMV, Beaudreau et al., 1960,Moscovici et al., 1969; AEV, Graf, 1975, Ishizaki and Shimizu, 1970). It appears that these viruses are defective and produce infectious progeny only with the aid of a replication-competent helper virus (Ishizaki et al., 1971; Moscovici 1975;Graf, 1976). These replication-defective leukemia viruses do not contain “sarc” related to the “sarc” sequenceof avian sarcoma viruses (Stehelin et al., 1975). REV, which is genetically unrelated to these viruses also lacks the “sarc” sequence of avian sarcoma viruses (Bishop and Bose, unpublished). Genetic control of transformation by these defective leukemia viruses is therefore probably unrelated to that of avian sarcoma viruses. The identification of the defect in oncogenic REV may prove valuable for determining the specific viral products responsible for leukemogenic transformation. ACKNOWLEDGMENTS This work was supported by Grant PCM77-25135 from the National Science Foundation and the National Institute of General Medical Sciences Grant GM-07126. REFERENCES BALUDA, M. A., and GOETZ,I. E. (1961). Morphological conversion of cell cultures by avian myeloblastosis virus. Virology 15, 185-199. BEARD, J. W. (1963). Oncornaviruses I: The avian tumor viruses. In “Ultrastructure of Animal Viruses and Bacteriophages” (A. J. Dalton and F. Haquenan, eds.), p. 261. Academic Press, New York. BEAUDREAU, G. S., BECKER, C., BONAR, R. A., WALLBANK, A. M., BEARD, D., and BEARD, J. W. (1960). Virus of avian myeloblastosis: XIV, Neoplastic response of normal chicken bone marrow treated with the virus in tissue culture. J. Nut. Cancer Inst. 24, 395-415. BISTER, K., HAYMAN, M. J., and VOGT, P. K. (1977). Defectiveness of avian myelocytomatosis virus MC29 Isolation of long-term nonproducer cultures
REPLICATION-DEFECTIVE and analysis of virus-specific polypeptide synthesis. 82, 431-448. CARPENTER, C. R., BOSE, H. R., and RUBIN, A. S. (197’7). Contact-mediated suppression of mitogeninduced responsiveness by spleen cells in reticuloendotheliosis virus-induced tumorigenesis. Cell. Virology
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CARPENTER, C. R., RUBIN, A. S., and BOSE, H. R. (1978). Suppression of mitogen-stimulated blastogenie response during reticuloendotheliosis virusinduced tumorigenesis: Investigations into the mechanism of action of the suppressor. J. Immuno2. 120, 1313-1320. FRANKLIN, R. B., KANG, C. Y., WAN, M. K., and BOSE, H. R. (1977). Transformation of chick embryo fibroblasts by reticuloendotheliosis virus. Virology 83, 313-321. FRANKLIN, R. B., MALDONADO,R. L., and BOSE,H. R. (1974). Isolation and characterization of reticuloendotheliosis virus transformed bone marrow ceils. Intervirology
3, 342-352.
GRAF, T. (1973) Two types of target cells for transformation with avian myelocytomatosis virus. Virology 54, 398-413. GRAF, T. (1975) In vitro transformation of chicken bone marrow cells with avian erythroblastosis virus. 2. Naturforsch. 3OC, 847-849. GRAF, T., ROYER-POKARA,B., SCHUBERT,G. T., and BENG, H. (1976). Evidence for the multiple oncogenic potential of cloned leukemia virus: In vitro and in tivo studies with avian erythroblastosis virus. Virology 71, 423-433. GROSS, L. (1970). “Oncogenic Viruses.” Pergamon, Oxford. HALPERN, M. S., WADE, E., RUCKER, E., BAXTERGRABBARD,K. L., LEVINE, A. S., and FRIIS, R. E. (1973). A study of the relationship of reticuloendotheliosis virus to the avian leukosis-sarcoma complex of virus. Virology 53, 287-299. HANAFUSA, H. (1977) In “Comprehensive Virology,” (H. L. Fraekel-Conrat and R. Wagner, eds.), Vol. 10, pp. 401-483. Plenum, New York. HARTELY, J. W., and ROWE,W. P. (1966). Production of altered cell foci in tissue culture by defective Moloney sarcoma virus particles. Proc. Naf. Acad. Sci. USA 55, 780-786.
ISHIZAKI, R., LANGLOIS, A. J., CHABOT, J., and BEARD, J. W. (1971). Component of strain MC29 avian leukosis virus with the property of defectiveness. J. Viral. 8, 821-827. ISHIZAKI, R., and SHIMIZU, T. (1970). Heterogeneity of strain R avian erythroblastosis virus. Cancer Res. 30, 2827-2831. KANG, C.-Y., and TEMIN, H. M. (1973). Lack of sequence homology among RNAs of avian leukosissarcoma viruses, reticuloendotheliosis viruses, and
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REV
29
chicken endogenous RNA-directed DNA polymerase activity. J. Viral. 12, 1314-1324. KANG, C.-Y., and TEMIN, H. M. (1974). Reticuloendotheliosis virus nucleic acid sequencesin cellular DNA. J. Viral. 14, 1179-1188. KANG, C.-Y., WONG,T. C., and HOLMES,K. V. (1975). Comparative ultrastructure study of four reticuloendotheliosis viruses. J. Viral. 16, 1027-1038. LANGLOIS,A. J., and BEARD, J. W. (1967). Convertedcell focus formation in culture by strain MC29 avian leukosis virus. Proc. Sot. Exp. Biol. Med. 126, 718-722. MALDONADO,R. L., and BOSE, H. R. (1973). Group specific antigen shared by the members of the reticuloendotheliosis virus complex. J. Viral. 17, 983-990. MALDONADO,R. L., and BOSE, H. R. (1975). Polypeptide and RNA composition of the reticuloendotheliosis virus complex. J. Viral. 17, 983-990. MOSCOVICI,C. (1967). A quantitative assay for avian myeloblastosis virus. Virology 68, 173-181. MOSCOVICI,C. (1975). Leukemic transformation with avian myeloblastosis virus: Present status. Curr. Top. Microbial. Immunol. 71, 79-101. MOSCOVICI,C. D., CHI, GAZZOLO,L., and MOSCOVICI, M. G. (1976). A study of plaque formation with avian RNA tumor viruses. Virology 73, 181-189. MOSCOVICI,D., MOSCOVICI,M. G., and ZANETTI, M. (1969). Transformation of chick embryo fibroblast cultures with avian myeloblastosis virus. J. Cell. Physiol. 73, 105-108. OLSON,L. D. (1967). Histopathological and hematologic changes in moribund stages of chicks infected with T-virus. Amer. J. Vet. Res. 28, 1501-1507. PURCHASE, H. G., and BURMESTER, B. R. (1972). In “Diseases of Poultry” (M. S. Hofsted, B. W. Calnek, C. F. Helmboldt, W. M. Reid, and H. W. Yoder, eds.), pp. 502-567. Iowa State Univ. Press, Ames, Iowa. PURCHASE,H. G., LUDFORD,C., NAZERIAN, K., and Cox, H. W. (1973). A new group of oncogenic viruses: Reticuloendotheliosis chick syncytial, duck infectious anemia, and spleen necrosis virus. J. Nut. Cancer Inst. 51, 489-499. PURCHASE, H. G., and WITTER, R. L. (1975). The reticuloendotheliosis viruses. In “Current Topics in Microbiology and Immunology” (P. K. Vogt, ed.), Vol. 71, pp. 103-124. Springer-Verlag, New York. ROSENBERG,H., BALTIMORE, D., and SCHER, C. D. (1975). In vitro transformation of lymphoid cells by Abelson murine leukemia virus. Proc. Nat. Acad. Sci. USA 72, 1932-1936. SCHER, C. D., and SIEGLER, R. (1975). Direct transformation of 3T3 cells by Abelson murine leukemia virus. Nature (London) 253, 729-731. SCOFIELD,V. L., andBosE, H. R., JR. (1978). Depression of mitogen response in spleen cells from reticulo-
30
HOELZER, FRANKLIN,
endotheliosis virus-infected chickens and their suppressive effect on normal lymphocyte response. J. Zmmunol. 120, 1321-1325. SEVOIAN, M. R., LAROSE, N., and CHAMBERLAIN, D. M. (1964). Avian lymphomatosis: VI, A virus of unusual potency and pathogenicity. Avian Dis. 8, 336-347. STEHELIN, D., GUNTAKA, R. V., VARMUS, H. E., and BISHOP, J. M. (1975). Purification of DNA complementary to nucleotide sequences required for neoplastic transformation of fibroblasts by avian sarcoma viruses. J. Mol. Bio!. 101, 349-365. TF,MIN, H. M., and KASSNER,V. K. (1974). Replication of reticuloendotheliosis virus in cell culture: Acute infection. J. Vkol. 13, 291-297. TEMIN, H. M., and KASSNER,V. K. (1975). Replication of reticuloendotheliosis virus in cell culture: Chronic infection. J. Gen. Viral. 27. 267-274.
AND BOSE
THEILEN, G. H., ZEIGEL, R. F., ~~~TWIEHAUS, M. J. (1966). Biological studies with RE virus (strain T) that induces reticuloendotheliosis in turkeys, chickens, and Japanese quail. J. Nat. Cancer Inst. 37, 731-743. VOGT,P. K. (1965).Avian tumor viruses. In “Advances in Virus Research” (K. M. Smith and A. Lauffer, eds.), Vol. 11, pp. 293-385. Academic Press, New York. WAITE, M. R., and ALLEN, P. T. (1975). RNA-directed DNA polymerase activity of reticuloendotheliosis virus: Characterization of the endogeneous and exogenous reactions. J. Vi/irol. 16, 872-879. WITTER, R. L., PURCHASE, H. G., and BURGOYNE, G. H. (1970). Peripheral nerve lesions similar to those of Marke’s disease in chickens inoculated with reticuloendotheliosis virus. J. Nnt. Cancer Inst. 45, 567-577.
93, 20-30
(1979)
Transformation by Reticuloendotheliosis Virus: Development of a Focus Assay and Isolation of a Nontransforming Virus JACALYN
D. HOELZER,’
Department
RAY B. FRANKLIN,
of Microbiology,
The University
AND
of Texas at Austin,
HENRY R. BOSE, JR. Austin,
Texas 7871i?
Accepted October 6, 1978
A focus assay for quantitating in vitro transformation by oncogenic reticuloendotheliosis virus (REV) has been developed in Japanese quail embryo fibroblast cultures. The titer of the transforming virus detected in the in vitro focus-forming assay correlates with the development of reticuloendotheliosis in chickens. The titration pattern of REV focus formation appears to follow two-hit kinetics. A nontransforming member has been identified in the REV stocks and is designated reticuloendotheliosis-associated virus (REV-A). The nontransforming virus, REV-A, is present in REV stocks in lOO-to lOOO-foldexcess over the transforming member. REV-A causes an initial cytopathic effect in chick and quail embryo fibroblast cultures. The surviving cells continue to divide leading to the development of a persistently infected culture. The persistently infected cultures are morphologically indistinguishable from uninfected avian fibroblast cultures. REV-A interferes with superinfection by the oncogenic member. The addition of REV-A to oncogenic preparations of REV increases focus formation and changes the titration pattern from two-hit to one-hit kinetics indicating that REV is defective.
occurs after a brief latent period (Temin and Kassner, 1974). Following this acute Members of the reticuloendotheliosis (RE) phase the cytopathic effect disappears and group of retroviruses have been isolated the cultures chronically yield virus (Temin from a number of avian species. These and Kassner, 1975). These persistently inviruses have similar morphology (Kang fected cultures are morphologically similar et al., 19’75),RNA and polypeptide composi- to uninfected fibroblasts. tion (Maldonado and Bose, 19751,a groupReticuloendotheliosis virus (REV), the reactive antigen (Maldonado and Bose, 19’761, prototype, is the only member presently and they share extensive RNA sequence included in the RE group shown to be capable homology (Kang and Temin, 1973). At the of oncogenic transformation. REV induces present time, members of the RE group can an acute systemic proliferative disease with be distinguished only on the basis of patho- distinctive lesions composed of reticulogenicity and cross-neutralization tests which endothelial cells in the liver, spleen, and reflect differences in virion surface glycopro- other visceral organs of experimentally teins (Purchase and Witter, 1975). These infected chickens (Sevoian et al., 1964; viruses induce a variety of syndromes Thielen et aZ., 1977; Olson, 1967). REVincluding spleen necrosis, anemia, visceral transformed cell lines have been isolated, reticuloendotheliosis, and lymphoid nerve cloned, and cultured from the bone marrow lesions (Purchase et al., 1973). When chick of REV-infected birds (Franklin et al., 1974). embryo fibroblast (CEF) cultures are in- The virus produced from these bone marrow fected with RE viruses, virus production cultures (BMC) transforms CEF cultures with the development of a cytopathic effect in vitro (Franklin et al., 1977). RNA tumor viruses are divided into ’ To whom reprint requests should be addressed. sarcoma and leukemia viruses based on their INTRODUCTION
0042-6822/79/030020-l1$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
20
REPLICATION-DEFECTIVE
oncogenic potential (Vogt, 1965). The leukemia viruses generally do not morphologically transform cells in culture. However, several viruses in addition to REV have been described which induce acute lymphoproliferative diseases and are capable of transforming cells in vitro. Avian myeloblastosis virus transforms bone marrow and yolk sac cultures (Beaudreau et al., 1960; Baluda and Goetz, 1961; Moscovici, 1967; Moscovici et al., 1975) and avian erythroblastosis virus strain R (AEV-R) (Ishizaski and Shimizu, 1970) and myelocytomatosis virus (MC29) transform CEF cultures (Langlois and Beard, 1967). Abelson mouse leukemia virus also transforms both bone marrow and mouse fibroblasts (Rosenberg et al., 1975; Scher and Siegler, 1975). These in vitro transforming leukemia viruses are replication defective (MC29, Ishizaki et al., 1971; AMV, Moscovici, 1975; AEV, Graf et al., 1976; Abelson, Scher and Siegler, 1975). In this paper we report the development of a transformation assay for reticuloendotheliosis virus. This assay permitted us to demonstrate that oncogenic stocks of REV are composed of a mixture of transforming and nontransforming viruses. MATERIALS
AND METHODS
Viruses. Unipore (Bio-Rad) filtered (0.4 pm) supernatant fluids from cultures of the REV-transformed chicken bone marrow cell line (BMC) harvested in log phase was the source of oncogenic REV used in these experiments. These REV preparations were free of particles containing avian leukosis gene sequences (Franklin et al., 1977). The plaque assay of Moscovici and co-workers (1976) has shown that this fluid contains 5 x 10” plaque-forming units (PFU)/ml. Assay in vivo revealed 1 x lo4 LD,,jml for l-day-old Hyline chickens. The nontransforming virus present in the oncogenic REV stock was prepared by infection of secondary SPAFAS chicken or Japanese quail embryo fibroblast cultures with a lop5 dilution of the oncogenic REV stock. The cells were subcultured once and culture fluid was harvested at 5 days after infection.
TRANSFORMING
REV
21
Cell cultures. The REV-transformed BMC line was maintained in RPM1 1640 medium supplemented with 8% heat-inactivated fetal bovine serum. These BMC cultures are free of mycoplasma and Marek’s disease virus antigens (R. L. Witter, personal communication). Fibroblast cultures were grown in RPM1 1640 supplemented with 10% tryptose phosphate broth and 5% heat-inactivated calf serum. Focus assay for REV on quail embryo jibroblast cultures. Primary quail embryo
fibroblast (QEF) cultures were subcultured into 60-mm tissue culture dishes (Falcon) at a density of 8 x lo5 cells/plate in 5 ml of RPM1 1640 with 10% tryptose phosphate broth and 1% heat-inactivated calf serum. After 4 hr the medium was removed. The cultures were infected with dilutions of the oncogenic REV preparation in 1 ml of medium containing 25 pglml DEAE-dextran. Duplicate plates were infected for each dilution tested. After a 1-hr adsorption period at 37”, each plate was overlayed with 7 ml of a 0.7% agar mixture containing Ham’s FlO with 0.7% Bacto-Agar, 5% heat-inactivated calf serum, 1% heat-inactivated chick serum, 1% beef embryo extract, 0.028% sodium bicarbonate, 700 U penicillin1600 U streptomycin, and 0.01 mg gentamycin. Five days after infection, a second agar overlay (2 ml) containing the same components was added. Foci were scored on the eighth day after infection. Isolation in the REV
qf the
nontransforming virus stock by end point dilution.
Secondary QEF or CEF cultures were infected with several dilutions from 10-l through lo+’ of the oncogenic REV stock according to the method described above. After adsorption, the virus inoculum was removed and replaced with medium containing 10% tryptose phosphate broth and 5% calf serum. Three days after infection each plate was subcultured into duplicate 60-mm plastic petri dishes. The medium was changed on the fourth day and the virus-containing culture fluids were harvested on the sixth day. The virus present at each dilution was assayed for transforming ability in QEF, and in l-day-old chickens as described previously (Bose and Levine, 1967).The number
22
HOELZER,
FRANKLIN,
AND BOSE
RESULTS of virus particles in each dilution was also detected by the reverse transcriptase Development of a Transformation Assay assay. for Reticuloendotheliosis Virus The standard exogenous reaction (Waite and Allen, 1975) was carried out in a final volume of 0.1 ml containing 100 mM NaCI, The replication of RE group viruses in 50 mM Tris-HCI (pH 8.3), 5 mM dithio- CEF cultures results in an initial acute threitol (DTT), 0.05 mM manganous acetate, cytopathic effect (Temin and Kassner, 1974). 7.5 piIf poly(rA), 1.9 @l4 oligo(dT) 12-18, This property has allowed the development 0.2% Triton x-100, and 20 #.IJi of [3H]dTTP of a quantitative plaque assay for RE group ([3HJthymidine triphosphate; 40 to 60 Ci/ viruses (Temin and Kassner, 1974;Moscovici mmol). Virus in the culture fluid was pelleted et al., 1976). This assay does not measure in a SW41 rotor for 45 min at 30,000 rpm. the transforming potential of the protoThe virus pellet was suspended in 0.2 ml type REV. buffer and lo-,ul virus samples were mixed REV transforms avian bone marrow and with the reaction mix and incubated at 3’7 spleen cells in vivo (Franklin 1974;Kang and for 1 hr. At the end of incubation 2 ml of Temin, 1974)and in vitro (Hoelzer and Bose, cold TP (0.4 M trichloracetic acid, 0.02 M unpublished). A continuous line of REVsodium pyrophosphate) was added to each transformed chicken bone marrow cells has tube. The precipitate was collected on been maintained for over 4 years in this 0.45~pm Millipore filters, washed, dried, laboratory. This line chronically releases and counted in Bray’s scintillation fluid. oncogenic REV which transforms CEF culVirus interference assay. A stock of the turesin vitro (Franklin et al., 1977).Efforts to nontransforming member of REV was used quantitate foci in CEF cultures have met to infect secondary QEF cultures. Stocks of with only limited success. Since MC29 prothe nontransforming virus were diluted duces much more distinct foci on QEF culfivefold since this virus induces an extensive tures than on CEF cultures (Bister et al., cytopathic effect in fibroblast cultures. The 1977), QEF cultures were tested to deterplates were subcultured on the third and mine whether a quantitative transformation sixth days after infection and then infected assay could be developed for REV. Subconwith an oncogenic REV preparation at a fluent secondary QEF cultures were infected multiplicity of appoximately five. Secondary with serial dilutions of REV. Small discrete uninfected QEF cultures were also infected foci of transformed cells appeared on QEF at this time with the oncogenic REV stock. cultures 8- 10 days after infection. The REV The medium was changed on the eighth day foci were composed of disoriented, densely and virus-containing culture fluids were packed, spindle-shaped cells rather than the harvested 10 days after infection. The trans- rounded cells observed in a Rous sarcoma forming virus in these cultures was quanti- virus focus. Fig. 1 is a photograph of the tated by the focus-forming assay described cellular morphology of a REV-transformed above. focus in a confluent monolayer culture Kinetics offocus formation of REV in the of QEF. presence of excess nontransforming virus. The titration of an oncogenic REV stock A series of dilutions of the oncogenic REV obtained from the BMC line is shown in stock obtained from the BMC line was made Fig. 2. At higher dilutions the number of and distributed to duplicate sets. A twofold foci falls proportionally with the square of dilution of the nontransforming virus was the dilution factor indicating two-hit kinetics added to one set in a ratio of 1:l. Medium (Hartely and Rowe, 1966). Numerous titrawas added to each dilution of the second set tions of culture fluid obtained from different in place of the nontransforming virus. These BMC clones ranged from lo3 to lo4 focussets of inocula were then used to infect QEF forming units (FFU)/ml. Titration of REV cultures for use in the REV focus-forming produced from several BMC clones in Hyline assay. chickens give an average LD,, of approxi-
REPLICATION-DEFECTIVE
FIG. 1. An REV focus formed on a confluent monolayer mixture 8 days after infection.
mately 104LD,dml. Therefore, there exists a correlation between the development of reticuloendotheliosis in birds and the transforming activity measured in this in vitro focus-forming assay.
TRANSFORMING
REV
of quail embryo fibroblasts
23
overlayed
with 0.74 ) agar
Isolation of a Nontransforming Oncogenic REV Stocks
Viru .s in
As shown in Fig. 2, the transform ning activity of REV in QEF cultures appea rs to
HOELZER,
24
FRANKLIN.
AND BOSE
REV preparations contain a mixture of transforming and nontransforming viruses. Included in the RE group are a number of viruses which fail to transform fibroblast cultures or induce neoplastic diseases in experimentally infected animals. In this group are Trager duck spleen necrosis virus, chick syncytial virus, and duck infectious anemia virus. At the present time we are not certain whether the nontransforming virus present in the oncogenic REV stocks is one of these previously identified agents. With this reservation, we will refer to the nontransforming virus as reticuloendotheliosisassociated virus (REV-A). . It
10-27.5
50
2 5 IO-37550 CONCENTRATION REV STOCK
I
I
25 OF
Characterization of the Nontransforming Virus in the Oncogenic REV Stock
IO-4
FIG. 2. The dose-response relationship between the concentration of REV and the number of transformed foci formed on QEF cultures. Each point represents the number of foci formed by infection with 1 ml of the oncogenic REV stock obtained from the BMC line at the indicated dilutions. These results were obtained from the average of duplicate cultures at 8 days after infection.
follow two-hit kinetics at higher dilutions. This suggests that the oncogenic stocks obtained from the BMC line contains a mixture of two viruses (Hartely and Rowe, 1966). To determine whether the oncogenic REV stock contains a mixture of two viruses, lo-fold dilutions were used to infect secondary CEF and QEF cultures. The infected plates were subcultured on the third day and the virus-containing culture fluids were harvested on the sixth day. The presence of virus in the culture fluid was detected by a reverse transcriptase assay. The results are shown in Table 1. Infectious virus was detected up to a lop7 dilution. This number correlates with the results obtained by Moscovici and co-workers (1976) of 5 x lo6 PFU/ml of BMC REV stock. The transforming activity of this stock has an average of 5 x lo4 FFU/ml. Since there are approximately lo7 infectious virus particles in the stock these results indicate that oncogenic
When the nontransforming virus (REV-A) is used to infect QEF cultures as extensive cytopathic effect develops. By the fifth day after infection over 80% of the cells have lysed. Those cells which survive this acute phase of infection recover, and this leads to the establishment of a persistently infected culture. These results are shown in Fig. 3. The persistently infected cultures are morphologically indistinguishable from uninfected quail embryo fibroblasts. As shown TABLE
1
REVERSE TRANSCRIPTASE ACTIVITY OF CEF CULTURES INFECTED WITH DILUTIONS OF AN ONCOGENIC REV STOCK
Dilution” 100 10-S 10-c 10-7 10-s Background
[SH]TdR triphosphate incorporation* 11,383 14,983 1,968 941 291 187
fl Dilution of oncogenic REV used to infect chicken embryo fibroblasts (1 ml/GO-mm plate). b Counts per minute of [3H]thymidine triphosphate in a lo-p1 sample obtained from a detergent-disrupted virus pellet from 10 ml of CEF culture fluid 5 days after infection.
REPLICATION-DEFECTIVE
TRANSFORMING
25
REV
The Nontransforming Member Interferes with Replication of the Oncogenic Virus
The ability of leukosis viruses to interfere with sarcoma viruses of the same subgroup is well documented and serves as a measure of genetic relatedness (Vogt, 1965). To determine whether the nontransforming virus in the oncogenic REV stock interferes with the replication of the transforming virus, QEF cultures were infected with REV-A. The infected cells were subcultured 3 days after infection and challenged with oncogenic REV 6 days after infection. An uninfected set of secondary QEF cultures was also infected with the oncogenic REV stock at that time. Five days after superinfection with the oncogenic REV, the virus0
12345678 DAY
9 AFTER
IO
INFECTION
FIG. 3. The cytopathic effect induced by the nontransforming virus (REV-A) on QEF cultures. Freshly transferred secondary QEF cultures (6.5 x lo5 cells/ 60-mm petri dish) were infected with 1 ml of REV-A stock at a dilution of loo (0) or 10-l (0) in the presence of 25 @g/ml DEAE-dextran. Control plates received only 25 pg/ml DEAE-dextran. After a 1-hr adsorption period the inocula were removed and 5 ml of medium with 10% tryptose phosphate and 5% calf serum was added to each plate. The cell numbers were determined each day for duplicate plates. After trypsinization the cells were counted with the use of a hemocytometer. The medium was changed each day on the remaining cultures. The results are expressed as percentage control for each day.
in Fig. 4, the persistently infected cultures chronically yield virus as detected by the reverse transcriptase assay. The virus released from the persistently infected cultures fails to transform either CEF or QEF cultures. The response of avian fibroblasts to this nontransforming virus present in the oncogenic REV stock is similar to that described for other RE group viruses (Temin and Kassner, 1974). When REV-A is injected into l-day-old chickens they fail to develop the hepatosplenomegaly characteristic of REV. The latent period becomes protracted and the birds develop an acute runting disease accompanied by paralysis (data not shown).
:a5 0”I 6
4
0
12345678 DAY
9 AFTER
10
INFECTION
FIG. 4. The time course for replication of REV-A is avian fibroblast cultures. Cultures were infected with REV-A and virus production was measured by the release of particles with reverse transcriptase activity. Medium was harvested each day from duplicate cultures infected with an REV-A stock at a dilution of lOa (0) or 10-l (0). Virus-containing culture fluid (10 ml) was pelleted and suspended in 0.2 ml buffer. Samples (10 $1 were tested for reverse transcriptase activity as described under Materials and Methods. The results are expressed as counts per minute of [3H]tymidine triphosphate incorporated into acid-precipitable material from lo-p1 samples.
HOELZER, FRANKLIN,
26
AND BOSE
TABLE 2 ESTABLISHMENT OF INTERFERENCE BY REV-A AGAINST ONCOGENIC REV
Dilution
Cultures infected with REV”
Cultures infected with REV-A and superinfected with REV”
10” 10-1 10-Z 10-3 10-d IO-” 10-e
TNTC’ TbiTC TNTC 28,19 1,3 010 090
14,lO 030 030 0 090 030 030
Cultures infected with REV-A o,o 090
(1Culture fluid from QEF cultures taken on the fifth day after infection with oncogenic REV (1 ml/60-mm plate). h Culture fluid from QEF cultures 10 days after infection with the nontransforming virus and 5 days after superinfection with oncogenic REV. ’ Too numerous to count.
containing culture fluids were harvested and titrated for focus-forming ability in QEF cultures. The results are shown in Table 2. Stocks obtained from the BMC line or from the oncogenic stock passaged one time in QEF cultures had titers of approximately lo4 FFU/ml. Cultures that were previously infected with the nononcogenic member produced approximately 10 foci at the highest concentration or 0.1% as many foci. These results indicate that the nontransforming virus interferes with the replication of oncogenic REV. Change in the Kinetics of REV Focus Formation with Addition of Excess Nontransforming REV
Since the oncogenic REV stocks contain both a transforming and a nontransforming virus we tested the hypothesis that the transforming virus was defective. Duplicate sets of QEF cultures were infected with serial dilutions of an oncogenic REV preparation. A fixed amount of REV-A was added to each dilution of the oncogenic stock and used to infect one set of plates. The duplicate set of cultures was infected with serial dilutions of the oncogenic stock to which no REV-A was added. These cultures were overlaid with agar and foci were counted 8 days after infection. As shown in Fig. 5, addition of REV-A increases the number of
foci formed. In the presence of excess nontransforming virus, focus formation by REV is converted from two-hit to single-hit kinetics. The conversion from two-hit to single-hit kinetics suggests that REV is replication defective and that the transformed foci are formed by the infection of neighboring cells by REV. Consistent with the hypothesis that oncogenic REV is replication defective, we have isolated clones of REV-transformed non-virus producing QEF. Oncogenic REV can be rescued from non-virus producing transformed cells by infection of these cultures with the nononcogenic members of the RE group. DISCUSSION
The RE group of avian retroviruses consists of four members of which only one causes leukemogenic transformation. REV, the prototype of the group, is able to transform cells present in the bone marrow and spleen of chicken and quail (Franklin et al., 1974, Kang and Temin, 19’74). REV also transforms chicken and bone marrow cells in vitro and these cells are morphologically identical to those isolated and subsequently cultured directly from infected birds (Hoelzer and Bose, unpublished). These bone marrow cell lines are therefore presumed to represent the target cell for REV.
REPLICATION-DEFECTIVE
I
10475
5.0
2.!j IO375 50 CONCENTRATION REV STOCK
2.5 OF
IO-’
FIG. 5. The dose-response relationship of REV focus formation on QEF cultures in the absence (0) and presence (0) of added REV-A. Each point represents the number of foci formed by 1 ml of oncogenic REV stock at each dilution. These results were obtained from the average of duplicate cultures 8 days after infection. Corrections were made for change in volume of inocula due to addition of REV-A or media at each dilution.
The virus produced by the bone marrow cell line in vitro is oncogenic in birds (Franklin et al., 19’74)and transforms CEF cultures (Franklin et al., 1977). In order to understand the mechanism of transformation by this acute leukemogenic virus, a quantitative transformation assay has been developed. Distinct foci appear on QEF cultures. A correlation exists between the quantitation of REV in QEF cultures determined by this focus-forming assay and the development of reticuloendotheliosis in chickens. Focus formation in these QEF cultures appears to follow two-hit kinetics at higher dilutions. This suggests that the development of foci depends on infection of the cell by more than one virus particle. If infection by two viruses is necessary for successful focus formation the chance of the event occurring will be dependent on the product of the chance of each event occurring alone. Thus the number of foci will fall proportionally with the square of the virus dilution. The apparent two-hit kinetics observed with REV in the QEF
TRANSFORMING
REV
27
focus assay can be explained two ways. Either transformation requires the expression of two viral genomes or, alternatively, the virus with transforming activity is replication defective. The oncogenic stocks of REV contains a mixture of transforming and nontransforming viruses. When REV is titrated in the presence of excess nontransforming virus the dose-response curve is converted from a two-hit to a single-hit pattern. This suggests that the two-hit kinetics of focus formation represents the requirement of dual infection of a cell by both the oncogenic and noncogenic viruses in order to produce a visible focus. Therefore the oncogenic virus is presumed to be replication defective but contains the genetic information required for transformation. Serial passage of oncogenic REV in CEF cultures quickly leads to attenuation of the oncogenic disease potential (Halpern et al., 1973; Witter et al., 1970). The apparent attenuation of REV may simply reflect the loss of the transforming virus from the stock. Since REV transforms cells of hematopoietic origin the attenuation of REV during serial propagation of CEF cultures may result because the target cell for transformation is not present. This may account for the failure of other laboratories to detect transformation by REV in fibroblast cultures (Temin and Kassner, 1974; Witter et al., 1970). The nontransforming virus, designated REV-A, is present in the oncogenic stock in a lOO- to lOOO-foldexcess. Replication of REV-A in avian fibroblast cultures leads to an initial acute cytopathic effect. The plaque assays described for REV by other laboratories (Moscovici, 1976; Temin and Kassner, 1974) were measuring the effect of the nontransforming virus present in the REV stock. Similar cytopathic effect appears in avian fibroblast cultures infected with other members of the RE group and has permitted the development of a plaque assay for these viruses as well (Temin and Kassner, 1974). Those cells which survive the acute phase divide and form a chronically infected culture. The persistently infected cultures are indistinguishable from uninfected fibroblast cultures. REV-A fails to induce reticuloendotheliosis when injected in chickens. The infected birds
28
HOELZER, FRANKLIN,
develop an acute runting disease and after a protracted latent period death occurs (Hoelzer and Bose, unpublished). When oncogenic stocks of REV are inoculated into line N and P chickens, line N chickens develop reticuloendotheliosis. By contrast, line P chickens fail to develop this oncogenic disease (Scofieldet al., 19’78).Line P chickens appear to restrict tumor development by the oncogenic REV member. Line P chickens, however, develop an acute runting disease similar to that observed with REV-A. In addition to providing a function(s) to permit the production of infectious REV, REV-A may also play a significant role in the pathogenesis induced by REV. REV is an extremely virulent retrovirus. The incubation period of REV may be as short as 5 days and the mortality of experimentally infected animals approaches 100% (Sevoian et al., 1964). Recently, we have reported that the ability of both T and B (Scofield and Bose, 1978; Carpenter et al., 1977) cells to respond to mitogens becomes cytostatically suppressed during the first 3 days after infection of chickens with oncogenic preparations of REV. This suppression can be transferred to splenic lymphocytes obtained from uninfected chickens with spleen cells from infected birds by a contact-mediated event involving a trypsin-sensitive surface protein (Carpenter et al., 1977). The suppressed T cells obtained from REV-infected birds fail to undergo cell division but are fully capable of cytotoxic effector function (Carpenter et al., 1978). The suppressor cells do not express REV antigens (Carpenter et al., 1978). It seemsreasonable to postulate that the rapid generation of the contactmediated cytostatic suppressor contributes to the extreme virulence of REV. The early appearance of the suppressor should prevent maximal T cell-mediated antitumor responses by limiting lymphoid cell proliferation. Infection of birds with REV-A, which replicates in the chicken but does not induce reticuloendotheliosis, induces this suppressor cell population (Carpenter et al., unpublished). Thus REV-A may contribute to the extreme virulence of REV by suppressing the host cell-mediated response. Acute avian leukemia viruses induce neo-
AND BOSE
plasms in vivo at a high rate in a short period of time. They cause myeloid and erythroid leukemias and some can induce fibrosarcomas (Beard, 1963; Gross, 1970; Purchase, 1972; Hanafusa, 1977). These avian viruses that induce leukemia have the capacity to transform cells of hematopoietic origin and fibroblasts in. vitro (MC29, Graf, 1973;AMV, Beaudreau et al., 1960,Moscovici et al., 1969; AEV, Graf, 1975, Ishizaki and Shimizu, 1970). It appears that these viruses are defective and produce infectious progeny only with the aid of a replication-competent helper virus (Ishizaki et al., 1971; Moscovici 1975;Graf, 1976). These replication-defective leukemia viruses do not contain “sarc” related to the “sarc” sequenceof avian sarcoma viruses (Stehelin et al., 1975). REV, which is genetically unrelated to these viruses also lacks the “sarc” sequence of avian sarcoma viruses (Bishop and Bose, unpublished). Genetic control of transformation by these defective leukemia viruses is therefore probably unrelated to that of avian sarcoma viruses. The identification of the defect in oncogenic REV may prove valuable for determining the specific viral products responsible for leukemogenic transformation. ACKNOWLEDGMENTS This work was supported by Grant PCM77-25135 from the National Science Foundation and the National Institute of General Medical Sciences Grant GM-07126. REFERENCES BALUDA, M. A., and GOETZ,I. E. (1961). Morphological conversion of cell cultures by avian myeloblastosis virus. Virology 15, 185-199. BEARD, J. W. (1963). Oncornaviruses I: The avian tumor viruses. In “Ultrastructure of Animal Viruses and Bacteriophages” (A. J. Dalton and F. Haquenan, eds.), p. 261. Academic Press, New York. BEAUDREAU, G. S., BECKER, C., BONAR, R. A., WALLBANK, A. M., BEARD, D., and BEARD, J. W. (1960). Virus of avian myeloblastosis: XIV, Neoplastic response of normal chicken bone marrow treated with the virus in tissue culture. J. Nut. Cancer Inst. 24, 395-415. BISTER, K., HAYMAN, M. J., and VOGT, P. K. (1977). Defectiveness of avian myelocytomatosis virus MC29 Isolation of long-term nonproducer cultures
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