VIROLOGY
67,
158-167
Potentiating
(1975)
Effect of lododeoxyuridine on MuLV in Mouse Embryo Fibroblastsl 0. NIWA,
Department
of Radiology,
A. DECLEVE,
Stanford
University Accepted
AND
School April
Replication
H. S. KAPLAN of Medicine,
Stanford,
California
94305
23, 1975
Treatment of mouse embryo fibroblasts with concentrations of 5-iododeoxyuridine (IUdR) too low to induce detectable replication of endogenous C-type viruses has been found to augment the susceptibility of permissive and nonpermissive cells to exogenous infection by murine leukemia viruses, without changing the kinetics of viral replication. No evidence could be obtained to support the hypothesis that this phenomenon results from induction of endogenous virus followed by complement&ion or recombination between endogenous and exogenous virus. Instead, the responsible mechanism seems likely to involve IUdR-induced DNA strand breakage, repair, and enhanced recombinational integration of the exogenous viral genome into cellular DNA. INTRODUCTION
nonestablished C57BL MEF cells by halogenated pyrimidine treatment have yielded either negative (Rowe and Hartley, 1972) or transient (Stephenson and Aaronson, 1972) responses. We now report that low concentrations of 5-iododeoxyuridine (IUdR) which are not capable of inducing detectable amounts of endogenous virus from C57BL MEF cells can significantly augment the susceptibility of permissive and nonpermissive cells to infection by exogenous RadLV, without changing the kinetics of viral replication. Experiments intended to elucidate the mechanism(s) involved are also described.
It is now well-established that the replication of endogenous C-type RNA viruses in nonproducer avian and mammalian cells may be induced by exposure to certain chemical and physical agents, of which the halogenated pyrimidines are the most potent (Weiss et al., 1971; Rowe et al., 1971; Lowy et al., 1971; Aaronson, Todaro, and Scolnick, 1971). C57BL mouse embryo fibroblast (MEF) cells have been reported to carry the murine leukemia virus genome integrated in DNA form (Gelb et al., 1973). The radiation leukemia virus (RadLV) is a leukemogenie endogenous B-tropic virus which is extractable from thymic lymphomas induced by X irradiation of C57BL mice (Lieberman and Kaplan, 1959; Kaplan, 1967). Recently, activation of an endogenous, B-tropic, C-type virus from an established cell line of C57BL MEF treated with 5- bromodeoxyuridine (BUdR) in vitro has been reported (Lieberman et al., 1973). Attempts to induce virus replication in
MATERIALS
Culture medium. Cultures were grown in Eagle’s minimal essential medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 units/ml), streptomycin (100 pg/ml), and polymixin (50 units/ml). Cells. Secondary mouse embryo fibroblast (BL-MEF) cultures were prepared from C57BWKa mice, as previously described (Decleve et al., 1970); BL-5, an established line of C57BL/Ka mouse embryo fibroblasts, has been previously de-
‘This investigation was supported by Grants CA 03352 and CA 10372 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. 158 Copyright@ 1975 All rights
by of reproduction
Academic Press,Inc. in any form reserved.
AND METHODS
NIWA,
DECLlhE
scribed, as has BL-5(RadL.V), a subline of BL-5 stably infected with RadLV (Lieberman et al., 1973); NIH/3T3 and normal rat kidney (NRK) cells, which were obtained from Dr. Stuart Aaronson, National Cancer Institute, Bethesda, Maryland, have been described (Aaronson and Stephenson, 1973; Due-Nguyen et al., 1966); rat XC cells (Klement et al., 1969) were kindly provided by Dr. A. J. Hackett, Naval Biomedical Research Laboratory, Oakland, California; CH(GLV) cells are an established cell line (C,H-1) of C&I-MEF stably infected with Gross-AKR MuLV (GLV) (Decleve et al., 1974). Virus preparations. RadLV serially passaged in vitro, and designated RadLV*, was harvested from the supernatant fluids of 3-day-old BL5(RadLV) cell cultures (Lieberman et al., 1973). GLV, serially passaged in oitro and designated GLV*, was harvested from the supernatant fluids of 3-day-old C&I(GLV) cell cultures (Declkve et al., 1974). Virus assays. XC assays were performed according to the X-ray modification previously described for adaptation of the direct XC plaque procedure to the assay of RadLV (Niwa et al., 1973). Immunofluorescence (IF) assays were carried out as previously described (Decleve et al., 1974). IUdR treatment. Plastic Petri dishes (60 mm) were seeded with 2 x lo6 BL-MEF or BL-5 cells. On the following day, the cells were treated with the indicated concentration of IUdR for 24 hr, after which the IUdR-containing medium was removed and the cells were washed and infected with virus, as previously described (Niwa et al., 1973; Decleve et al., 1974). Two days after infection, the cells were overlaid with 2 x lo6 MEF cells in order to compensate for the cell killing effect of IUdR, and the cultures were incubated for 2 more days. At that time, the cells were X-irradiated with 5000 R, and 1 x lo8 XC cells were seeded on the dishes. Five days later, the dishes were fixed, and the number of plaques scored. When an IF assay was performed, the secondary overlay and X irradiation were
AND
159
KAPLAN
omitted. The cells were simply trypsinized and processed for indirect immunofluorescence 4 days after viral infection. Whenever necessary, the cell cultures were handled under special illumination (“Bug Lite,” General Electric Company), which emits no wavelengths shorter than 500 nm, in order to avoid the photodynamic action of visible light on IUdR (Wacker, 1963). Virus irradiation. Virus was diluted in phosphate-buffered saline (PBS). Virus samples were placed in watch dishes and gently swirled at 4” during UV irradiation with a germicidal lamp at 16.5 ergs/mm’/ sec. For X-ray irradiation, the virus samples were irradiated in plastic dishes with a 50 kVp twin beryllium window tube X-ray unit (Loevinger and Huisman, 1965) at a dose rate of 0.14 kradlsec. Cell irradiation. Twenty-four hours after cell seeding, medium was replaced with 2 ml of PBS. UV irradiation was performed at room temperature at a dose rate of 2.1 ergs/mm “/sec. RESULTS
Effect of IUdR on cell growth and plaque titer. BL-MEF cells were seeded onto
plates, together with varying concentrations of IUdR. After 24 hr, the IUdR-containing medium was removed, and the cells were rinsed with fresh medium. Cell growth was monitored daily thereafter. The doubling time of control cells under these conditions was approximately 24 hr in all experiments. IUdR has a marked depressing effect on the growth of BL-MEF cells (Fig. 1). It has been reported that RadLV replicates better in cells in log phase growth than in those in stationary phase (Niwa et al., 1973; Decleve et al., 1974). Therefore, the cell growth inhibitory effect of IUdR would have been expected to decrease the number of plaques observed. This expectation was not confirmed in an experiment in which cells were treated with 4 pg/ml of IUdR for 24 hr 1 day after seeding in order to avoid the lag phase. Then serial dilutions of a RadLV* preparation were assayed. Although titration patterns of the
160
EFFECT
OF
IODODEOXYURIDINE
IN
virus were of the one-hit type in both IUdR-treated and control cells, the virus titer was approximately twofold greater in the IUdR-treated cells (Fig. 2). Influence of IUdR concentration on virus titer. C57BL MEF cells were treated with various concentrations of IUdR, and virus dilutions (lo-’ to 10e5) were assayed by the XC test. IUdR alone at concentrations up to 100 pg/ml did not induce detectable amounts of endogenous virus from BLMEF cells in this short-term assay (Fig. 3). However, pretreatment of cells with IUdR markedly increased the number of plaque-
MOUSE
a d@z
EMBRYO
$-o-011•-0110-d
-
4t 0
L, 0
.,
1 1
FIBROBLASTS
1
10
IUdr OR Tdr CONCENTRATION bra/ml) I I ’ 1 10 100 lUdr CONCENTRATION (uM) I 10 100 Tdr CONCENTRATION WI
100
J I
FIG. 3. Influence of IUdR and TdR concentration on virus titer as detected by plaque assay. O---O = IUdR alone; B---W = TdR + virus; 04, 04, 04 = replicate experiments with IUdR + virus. RadLV* was used at concentrations of lo-’ and 10e5.
0
1
2
3
4
5
6
7
DAYS
FIG. 1. The effect of various doses of IUdR on C57BL MEF cell growth. 0 = 0 pg; 0 = 1 pg; n = 2 pg; 0 = 5 pg; A = 10 pg; A = 2 ag; 0 = 50 Pg.
10-3
10-5
10-4 VIRUS
DILUTION
FIG. 2. Titration curve of RadLV* on IUdR-treated cells (O--O) and control cells (04).
forming cells observed after infection with exogenous RadLV. The optimal concentration of IUdR for enhancing plaque yield was lo-40 PLM (4-10 pg/ml). In this concentration range, virus titer was increased nearly threefold, as compared with the control value. Higher doses of IUdR were less effective, possibly because the toxicity of IUdR overrides the enhancing effect. Since IUdR alone did not yield any plaques under these experimental conditions, the higher number of plaques found in cultures pretreated with IUdR cannot be ascribed to simple additive effects of exogenous virus and infectious endogenous virus induced by IUdR. Instead, the effect of IUdR on virus titer apparently involves the potentiation of cell response to infection by exogenous virus. Augmentation of infectivity of exogenous virus by IUdR pretreatment of indicator cells was also observed using the IF assay (data not shown). However, the increase in titer in this experiment was somewhat higher than that observed by the XC test. The potentiating effect is again observed at all virus concentrations by IF, and the titration curves are of the one-hit type for both the IUdR-treated cultures and the controls. Although the proportion of cells infected
NIWA.
DECLIhE
by exogenous RadLV was increased by IUdR pretreatment, indicating an increased susceptibility of the treated cells to infection, the yield of progeny virus per infected cell, as measured by titration of the supernatant fluids, was paradoxically usually somewhat decreased relative to that of control cultures (data not shown). This reduced yield of infectious virus was probably a secondary consequence of the inhibitory influence of IUdR on cell growth (Fig. l), since it has been shown that RadLV replication is closely coupled to cell replication (Declbve et al., 1975). Lack of effect of X-ray irradiation and secondary overlay on the potentiating effect of IUdR. X-ray irradiation of RadLV*-
infected test cells is a routine step in the modified XC assay used in this laboratory; it helps visualize plaques more clearly without decreasing the sensitivity of the assay, as does UV irradiation (Niwa et al., 1973). Since the halogenated pyrimidines are known to be effective radiosensitizers (Djordjevic and Szybalski, 1960; Kaplan, Smith, and Tomlin, 1962), it seemed important to determine whether the potentiating effect observed after IUdR treatment might be due to the synergism of IUdR and X irradiation. The data of Table 1 indicate that an X-ray dose of 5000 R did not influence the titer of exogenous virus in cells pretreated with IUdR. Moreover, the potentiating effect of IUdR was also obTABLE
1
LACK OF EFFECT OF X-RAY ON THE POTENTIATING ACTION OF IUdRa IUdR (rd
No. plaques
(5Z&
0
+ -
0.25
+ -
1
+ -
4
+ -
16
+ -
64
+ -
n RadLV*
was used at a lo-’
16 14 26 26 33 30 42 44 35 31 33 32 dilution.
AND
KAPLAN
161
served with the IF assay, which does not include an irradiation step. In all of the experiments reported here, a secondary overlay with BL-MEF cells was routinely used to compensate for the cell killing effect of IUdR (Fig. 1). The use of a secondary overlay has been reported to improve the sensitivity of the UV-XC cell test (Lowy et al., 1971; Teich et al., 1973). Several replicate experiments comparing plaque production with and without the use of a secondary overlay revealed that the overlay technique did not show any selectivity for IUdR-damaged cultures. Moreover, potentiation by IUdR was also seen with the IF assay, which does not involve a secondary overlay step. It is therefore concluded that neither X irradiation nor the secondary overlay procedure contributed to the potentiation response. Competitive inhibition by thymidine of the potentiating effect of IUdR. It has been
shown that the incorporation of IUdR into host-cell DNA is necessary for IUdR activation of murine leukemia virus (Teich et al., 1973). We therefore tested whether analog incorporation into host-cell DNA is also required for the potentiating action of IUdR on cell susceptibility to exogenous virus. Addition of an equal concentration of thymidine (TdR), a natural precursor of DNA synthesis which is a known competitive inhibitor of IUdR incorporation into DNA, completely blocks the potentiating effect of IUdR on relative plaque yield (Table 2). An equimolar concentration of TdR alone had no effect on exogenous virus titer (Fig. 3). Target size analysis of RadLV* on the IUdR pretreated cells. One possible mech-
anism of potentiation by IUdR postulates the complementation of defective exogenous virus particles by endogenous virus partially activated by IUdR. If this were so, the amount of genetic information required by exogenous virus in IUdR-treated cells should be less than that required in control cells. Inactivation of RadLV* by UV light was therefore studied to determine whether differences in viral genome target size are observed concomitantly with the potentiating effect of IUdR. The virus was irradiated with graded doses of UV light and
162
EFFECT
OF
IODODEOXYURIDINE
IN
assayed on control cells or on IUdR-(5 pg/ml) pretreated cells. Both inactivation curves were essentially exponential (with slight shoulders) and virtually identical (Fig. 4). The dose (D,,) required to inactivate RadLV to 37% of the initial titer was 745 ergs/mm’, which is comparable with values obtained for other MuLV (Kelloff et al., 1970; Nomura et al., 1972). Thus, the amount of viral genetic information required for RadLV* replication was not significantly less in IUdR-pretreated than in control cells. The same conclusion was reached from the study of X-ray survival curves of the virus on IUdR-treated and control cells (data not shown). Host range analysis of the progeny virus IUdR pretreatment of cells. If IUdR
after
potentiation
were the result of recombinaTABLE
COMPETITIVE
INHIBITION
2
OF IUdR
POTENTIATION
BY THYMIDINE
IUdR
TdR
0
0 0 6&ml
6rg/ml 6 &ml
100
I
Relative plaque number 100
170 loo
I
I
-
x
10-l
-
h
MOUSE
EMBRYO
FIBROBLASTS
tion of defective exogenous virus particles with partially activated endogenous virus, the hybrid viral progeny might be expected to differ from the input virus with respect to such attributes as host range. Accordingly, progeny virus from IUdR-pretreated, virus-infected cells was studied for its host range pattern. BL-5 cells are known to harbor B-tropic virus (Lieberman et al., 1973). Control and IUdR-pretreated BL-5 cells were infected with either RadLV* or GLV*. The tissue culture fluids taken 4 days after IUdR treatment were titered on BL-5, NIH-3T3, and NRK cells. IUdR pretreatment did not change the host range of RadLV* or GLV*, despite the fact that the latter was passaged once on BL-5 (Fv-lb) cells (Table 3). Potentiating effect of IUdR on exogenous virus replication in nonpermissive cells and in rat cells. It has been reported that GLV*
(an N-tropic virus), when titered on BLMEF (Fv-lb) cells, shows a two-hit titration pattern (Decleve et al., 1975). It seemed of interest to determine whether IUdR pretreatment of nonpermissive cells influences viral kinetics as well as susceptibility to infection. IUdR pretreatment of BL-MEF cells increased their sensitivity to GLV* by twofold, but the two-hit titration pattern of the virus remained unchanged (data not shown). Even though their efficiency of infection is low, both N-tropic and B-tropic MuLV can replicate on rat (NRK) cells (Decleve et al., 1975). An experiment was performed to test whether IUdR pretreatment (at 5 pg/ml) can also potentiate MuLV infection of NRK cells. As shown in Fig. 5, IUdR increased the plaque titer of GLV* on NRK cells by about twofold. Lack of potentiating irradiation. UV irradiation
10‘3
-
10-4
. 0
I 100 IRRADIATION
FIG.
measured (@A)
4. UV inactivation on IUdR-pretreated C57BL MEF.
I
I
200
300
TIME
curves (O--O)
(SEC)
of RadLV* as and control
effect
of
UV
of cells was studied for its possible potentiating effect on virus replication. BL-MEF cells were irradiated with UV (2.1 ergs/mm*/sec) at 24 hr after seeding and immediately thereafter infected with RadLV. Four days after infection, no potentiating effect was found by the XC assay (Fig. 6). Instead, there was a sigmoid inactivation of the susceptibility of cells to virus replication as a function of UV dose.
NIWA,
DECLEVE TABLE
INFLUENCE
OF IUdR
AND
163
KAPLAN
3
PRETREATMENT ON HOST-RANGE OF PROGENY VIRUS IN SUPEHNATANTS CULTURES INFECTED WITH RadLV* OR GLV*a Source of supernatant
Plaque
Supernatant dilution
no. on various
BL-5 No IUdR
RadLV* BL-5
infected cultures
GLV’ infected BL-5 cultures
IUdR
pretreated
RadLV* BL-5
infected cultures
GLV* infected BL-5 cultures
0 NOTE: from mice homozygous
TMTC TMTC 219 63 1 0
l/5 l/50 l/500 l/5 l/50 l/500
TMTC TMTC 37 57 2 0
NIH
CELL
test cells
3T3
NRK
0 0 0
0 0 0 0 0 0
TMTC TMTC 216 0 0 0
0 0 0 0 0 0
TMTC TMTC 248
RadLV* is a B-tropic murine leukemia virus, whereas GLV* is N-tropic. BL-5 cells are derived of strain C57BL, homozygous for Fv-I*; NIH 3T3 cells are derived from NIH Swiss mice, which are for Fv-1”; NRK cells are rat cells used to detect the possible induction of xenotropic virus.
11
10-z I 0 4-l
4-2 VIRUS
FIG. treated cells.
l/5 l/50 l/500 115 l/50 l/500
OF BL-5
5. Titration (O-48
4-3
DILUTIONS
curves of GLV* and control (O--O)
on IUdR-prerat (NRK)
DISCUSSION
These experiments have revealed a twoto threefold increase in cellular susceptibility to MuLV infection, as measured by IF-positive or plaque-forming cell number, in IUdR-pretreated cells infected by RadLV* or GLV*, without a concomitant change in viral replication kinetics or in the yield of infectious virus. Two possible explanations for the potentiating action of the halogenated pyrimidines, which may
10
20
IRRADIATION
FIG. 6. Effect of UV irradiation ity to virus infection.
30
40
TIME (SEC)
on cell susceptibil-
also be relevant to the enhancement of XC cell DNA transfection in BUdR-pretreated chicken fibroblasts (Svoboda et al., 19’73), have been considered: (1) induction of endogenous virus by the halogenated pyrimidine followed by complementation or recombination between endogenous and defective exogenous virus; and (21 enhancement of exogenous virus replication as a result of IUdR-induced DNA strand breakage, repair, and recombinational integration of the viral genome into cellular DNA.
164
EFFECT
OF IODODEOXYURIDINE
Animal virus preparations often contain defective particles carrying incomplete or damaged viral genomes. It is probable that our RadLV* and GLV* preparations similarly contain defective particles which cannot replicate unaided, even in permissive cells. If pretreatment of cells with IUdR partially activates an endogenous MuLV genome, the activated virus may rescue or may be rescued by the exogenous defective MuLV, resulting in an increased yield of virus of heterogeneous derivation. The interaction of exogenous Rous sarcoma virus with endogenous helper virus has been reported in chicken cells (Hanafusa et al., 1970; Hanafusa et al., 1972). However, several ts mutants of MuLV have been isolated which replicate stably at the permissive temperature without losing their ts character (Stephenson et al., 1972; Wong et al., 1973). One of these mutants, which also replicates poorly at the nonpermissive temperature, nevertheless yields progeny virus which retains its ts character. Thus, it would appear that recombination does not readily occur between endogenous and exogenous murine leukemia viruses. Evidence for the partial activation of C-type viruses also exists. Leukemia virus group-specific antigen appeared in BUdRtreated rat embryo cells in the absence of detectable infectious virus (Freeman et al., 1973). Group-specific antigens were also detected after short-term culture of thymocytes of various strains; again, infectious virus could not be detected (Lonai et al., 1974). Other studies on the spontaneous expression of C-type RNA virus genes detected by the appearance of group-specific antigen (Dougherty and Di Stefano, 1966; Heubner et al., 1970; Hanafusa et al., 1973; Parks et al., 1973) or of virus-specific RNA (Hayward and Hanafusa, 1973; Benveniste et al., 1973; Fan and Baltimore, 1973; Okabe et a1.,1973) in the absence of infectious virus also appear to involve partial activation. If the endogenous virus genome is partially activated by IUdR and requires complementation by defective exogenous virus for its complete expression, the amount of genetic information required for the exoge-
IN MOUSE
EMBRYO
FIBROBLASTS
nous virus to replicate should be less than that under normal conditions. This should be reflected in the relative target size of exogenous virus; the target size of the virus for UV irradiation should be smaller in the IUdR-pretreated cells than that in the control cells. A precedent exists in the case of bacteriophage in the well-known phenomenon of prophage reactivation (Jacob and Wollman, 1953; Blanc0 and Devoret, 1973). The relative target size of X phage is much smaller when the host cell is lysogenie for a homologous heteroimmune prophage. Prophage reactivation has also been observed with phage P, (Bertani and Bertani, 1971). The result shown in Fig. 4 seems to eliminate this possibility within the resolution of our methods. Host-range studies of progeny virus from IUdR-pretreated RadLV* - or GLV*-infected cells suggest that the progeny virus retained the original characteristics of the input exogenous virus. These experiments thus provide no evidence in support of the hypothesis that the progeny virus may represent recombinants of endogenous and exogenous virus (assuming that the assay methods used were sensitive enough to detect possible host-range variations). The alternative hypothesis involves the following considerations: type-C virus replication is now known to require the reverse transcription of viral RNA to the DNA form (Baltimore, 1970; Temin and Mizutani, 1970), followed by its integration into host-cell DNA (Balduzzi, 1973; Varmus et al., 1973; Markham and Baluda, 1973). The process of viral genome integration is thought to be through recombination between viral and host DNA, which requires the transient production of single-strand breaks in host DNA (Leis et al., 1973). IUdR is known to be a radiosensitizer for both UV and X-ray irradiation (Djordevic and Szybalski, 1960; Kaplan et al., 1962), creating single-strand breaks in cellular DNA where it replaces TdR (Wacker, 1963; Hotz and Walzer, 1970). It is therefore possible that IUdR enhances the integration of viral DNA into host-cell DNA by creating single-strand breaks in host-cell DNA, thus increasing the probability of
NIWA,
DECLEVE
viral DNA recombination with host-cell DNA. It is of interest that a two- to fourfold increase in the efficiency of transformation of mouse 3T3 cells by SV40, which also must integrate its genome into host-cell DNA, resulted when they were pretreated with IUdR or BUdR (Todaro and Green, 1964). Thus, although no direct evidence that IUdR enhances viral integration was provided by our experiments, the potentiating effect of halogenated pyrimidines on cellular susceptibility to infection by these viruses seems most plausibly attributed to such a mechanism. Moreover, whereas the photochemical reactions of halogenated pyrimidines in DNA lead to strand breaks, pyrimidine dimers are the major type of lesion in DNA damaged by UV irradiation. The fact that UV irradiation did not show any potentiating effect on cell susceptibility to MuLV infection suggests that the specific type of damage sustained by the host cell DNA may be an important determinant of the potentiation phenomenon. One-hit kinetics of UV inactivation of RadLV* is consistent with data reported for other C-type RNA viruses (Kelloff et al., 1970; Nomura et al., 1972; Friis, 1971; Levinson and Rubin, 1966; Rubin and Temin, 1959). The 60-70s RNA genome of C-type RNA viruses consists of 30-40s RNA subunits (Montagnier et al., 1969; Canaani et al., 1973). Biochemical studies of murine C-type virus replication suggest that these subunits are not redundant (Fan and Baltimore, 1973). The observed first order kinetics of virus inactivation by UV radiation would also be consistent with the absence of redundancy. REFERENCES AARONSON, S. A., and STEPHENSON, J. R. (1973). Independent segregation of loci for activation of biologically distinguishable RNA C-type viruses in mouse cells. Proc. Nut. Acad. Sci. USA 70, 2055-2058. AARONSON, S. A., TODARO, G. J., and SCOLNICK, E. M. (1971). Induction of murine C-type viruses from clonal lines of virus-free BALB/3T3 cells. Science 174, 157-159. BALDUZZI, P. C. (1973). Mechanism of oncogenic transformation by Rous sarcoma virus. III. Role of
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IODODEOXYURIDINE
Acad. Sci. USA 70, 2415-2419. FRIIS, R. R. (1971). Inactivation of avian sarcoma virus with UV light: a difference between helperdependent and helper-independent strains. Virology 43, 521-523. GELB, L. D., MILSTIEN, J. B., MARTIN, M. A., and AARONSON, S. A. (1973). Characterization of murine leukaemia virus-specific DNA present in normal mouse cells. Nature New Biol. 244, 76-79. HANAFUSA, H., AOKI, T., KAWAI, S., MNAMOTO, T., and WILSNACK, R. E. (1973). Presence of antigen common to avian tumor viral envelope antigen in normal chick embryo cells. Virology 56, 22-32. HANAFUSA, T., HANAFUSA, H., MNAMOTO, T., and FLEISSNER, E. (1972). Existence and expression of tumor virus gene in chick embryo cells. Virology 47, 475-482. HANAFUSA, H., MIYAMOTO, T., and HANAFUSA, T. (1970). A cell-associated factor essential for formation of an infectious form of Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 66, 314-321. HAYWARD, W. S., and HANAFUSA, H. (1973). Detection of avian tumor virus RNA in uninfected chicken embryo cells. J. Virol. 11, 157-167. HOTZ, G., and WALSER, R. (1970). On the mechanism of radiosensitization by 5-bromouracil. The occurrence of DNA strand breaks in UV-irradiated phage T4 as influenced by cysteamine. Photochem. Photobiol. 12, 207-218. HUEBNER, R. J., KELLOFF, G. J., SARMA, P. S., LANE, W. T., TURNER, H. C., GILDEN, R. V., OROSZLAN, S., MEIER, H., MYERS, D. D., and PETERS, R. L. (1970). Group-specific antigen expression during embryogenesis of the genome of the C-type RNA tumor virus: implications for ontogenesis and oncogenesis. Proc. Nat. Acad. Sci. USA 67, 366-376. JACOB, F., and WOLLMAN, E. L. (1953). Induction of phage development in lysogenic bacteria. Cold Spring Harbor Symp. Qua&. Biol. 18, 101-121. KAPLAN, H. S. (1967). On the natural history of the murine leukemias: presidential address. Cancer Res. 27, 1325-1340. KAPLAN, H. S., SMITH, K. C., and TOMLIN, P. A. (1962). Effect of halogenated pyrimidines on radiosensitivity of E. coli. Radiat. Res. 16, 98-113. KELLOFF, G., AARONSON, S. A., and GILDEN, R. V. (1970). Inactivation of murine sarcoma and leukemia viruses by ultraviolet irradiation. Virology 42, 1133-1135. KLEMENT, V., ROWE, W. P., HARTLEY, J. W., and PUGH, W. E. (1969). Mixed culture cytopathogenicity: a new test for growth of murine leukemia viruses in tissue culture. Proc. Nat. Acad. Sci.
USA 63.753-758. LEIS, J. P., BERKOWER, I., and HURWITZ, J. (1973). Mechanism of action of ribonuclease H isolated from avian myeloblastosis virus and Escherichia coli. Proc. Nat. Acad. Sci. USA 70, 466-470.
IN
MOUSE
EMBRYO
FIBROBLASTS
LEVINSON, W., and RUBIN, H. (1966). Radiation studies of avian tumor viruses and Newcastle disease virus. Virology 28, 535-542. LIEBERMAN, M. L., and KAPLAN, H. S. (1959). Leukemogenic activity of filtrates from radiation-induced lymphoid tumors of mice. Science 130, 387-388. LIEBERMAN, M., NIWA, O., DECL%VE, A., and KAPLAN, H. S. (1973). Continuous propagation of radiation leukemia virus on a C57BL mouse embryo fibroblast line, with attenuation of leukemogenic activity. Proc. Nat. Acad. Sci. USA 70, 1250-1253. LOEVINCER R., and HUISMAN, P. A. (1965). A twintube 50-kVp beryllium-window X-ray unit for microbial radiobiology. Radiat. Res. 24, 357-367. LONAI, P., DECL~VE, A., and KAPLAN, H. S. (1974). Spontaneous induction of endogenous murine leukemia virus-related antigen expression during short-term in uitro incubation of mouse lymphocytes. Proc. Nat. Acad. Sci. USA 71, 2008-2012. LOWU, D. R., ROWE, W. P., TEICH, N., and HARTLEY, J. W. (1971). Murine leukemia virus: high-frequency activation in uiuo by 5-iododeoxyuridine and 5-bromodeoxyuridine. Science 174, 155-156. MARKHAM, P. D., and BALUDA, M. A. (1973). Integrated state of oncornavirus DNA in normal chicken cells and in cells transformed by avian myeloblastosis virus. J. Viral. 12, 721-732. MONTAGNIER, L., GOLD@ A., and VIGIER, P. (1969). A possible subunit structure of Rous sarcoma virus RNA. J. Gen. Viral. 4, 449-452. NIWA, O., DECL~VE, A., LIEBERMAN, M., and KAPLAN, H. S. (1973). Adaptation of plaque assay methods to the in vitro quantitation of the radiation leukemia virus. J. Viral. 12, 68-73. NOMURA, S. BASSIN, R. H., TURNER, W., HAAPALA, D. K., and FISCHINGER, P. J. (1972). Ultraviolet inactivation of Moloney leukemia virus: relative target size required for virus replication and rescue of “defective” murine sarcoma virus. J. Gen. Viral. 14, 213-217. OKABE, H., GILDEN, R. V., and HATANAKA, M. (1973). RD 114 virus-specific sequences in feline cellular RNA: detection and characterization. J. Virol. 12, 984-994. PARKS, W. P., LIVINGSTON, D. M., TODARO, G. J., BENVENISTE, R. E., and SCOLNICK, E. M. (1973). Radioimmunoassay of mammalian type C viral protein. III. Detection of viral antigen in normal murine cells and tissues. J. Exp. Med. 137,622-635. ROWE, W. P., and HARTLEY, J. W. (1972). Studies of genetic transmission of murine leukemia virus by AKR mice. II. Crosses with Fu-1 strains of mice. J. Exp. Med. 136, 1286-1301. ROWE, W. P., HARTLEY, J. W., LANDER, M. R., PUGH, W. E., and TEICH, N. (1971). Noninfectious AKR mouse embryo cell lines in which each cell has the capacity to be activated to produce infectious murine leukemia virus. Virology 46, 866-876.
NIWA,
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RUBIN, H., and TEMIN, H. M. (1959). A radiological study of cell-virus interaction in the Rous sarcoma. Virology 7, 75-91. STEPHENSON, J. R., and AARONSON, S. A. (1972). Genetic factors influencing C-type RNA virus induction. J. Erp. Med. 136, 175-184. STEPHENSON, J. R., REYNOLDS, R. K., and AARONSON, S. A. (1972). Isolation of temperature sensitive mutants of murine leukemia virus. Virology 48, 749-756. SVOBODA, J., HLO~ANEK, I., MACH, O., MICHLOVA, A. RIMAN, J., and URBANKOVA, M. (1973). Transfection of chicken fihroblasts with single exposure to DNA from virogenic mammalian cells. J. Gen. Viral. 21, 47-55. TEICH, N., Lowv, D. R., HARTLEY, J. W., and ROWE, W. P. (1973). Studies of the mechanism of induction of infectious murine leukemia virus from AKR mouse embryo cell lines by 5-iododeoxyuridine and 5-bromodeoxyuridine. Virology 51, 163-173.
AND
KAPLAN
167
TEMIN, H. M., and MIZUTANI, S. (1970). RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature (London) 226, 1211-1213. TODARO, G. J., and GREEN H. (1964). Enhancement by thymidine analogs of susceptibility of cells to transformation by SV40. Virology 24, 393-400. VARMUS, H. E., VOGT, P. K., and BISHOP, J. M. (1973). Integration of deoxyribonucleic acid specific for Rous sarcoma virus after infection of permissive and nonpermissive hosts. Proc. Nut. Acad. Sci. USA 70, 3067-3071. WACKER, A. (1963). Molecular mechanism of radiation effects. hog. Nucleic Acid Res. 1, 369-398. WEISS, R. A., FRIIS, R. R., KATZ, E., and VOGT, P. K. (1971). Induction of avian tumor viruses in normal cells by physical and chemical carcinogens. Virology 46, 920-938. WONG, P. K. Y., and MCCARTER, J. A. (1973). Genetic studies of temperature-sensitive mutants of Moloney-murine leukemia virus. Virology 53,319-326.
67,
158-167
Potentiating
(1975)
Effect of lododeoxyuridine on MuLV in Mouse Embryo Fibroblastsl 0. NIWA,
Department
of Radiology,
A. DECLEVE,
Stanford
University Accepted
AND
School April
Replication
H. S. KAPLAN of Medicine,
Stanford,
California
94305
23, 1975
Treatment of mouse embryo fibroblasts with concentrations of 5-iododeoxyuridine (IUdR) too low to induce detectable replication of endogenous C-type viruses has been found to augment the susceptibility of permissive and nonpermissive cells to exogenous infection by murine leukemia viruses, without changing the kinetics of viral replication. No evidence could be obtained to support the hypothesis that this phenomenon results from induction of endogenous virus followed by complement&ion or recombination between endogenous and exogenous virus. Instead, the responsible mechanism seems likely to involve IUdR-induced DNA strand breakage, repair, and enhanced recombinational integration of the exogenous viral genome into cellular DNA. INTRODUCTION
nonestablished C57BL MEF cells by halogenated pyrimidine treatment have yielded either negative (Rowe and Hartley, 1972) or transient (Stephenson and Aaronson, 1972) responses. We now report that low concentrations of 5-iododeoxyuridine (IUdR) which are not capable of inducing detectable amounts of endogenous virus from C57BL MEF cells can significantly augment the susceptibility of permissive and nonpermissive cells to infection by exogenous RadLV, without changing the kinetics of viral replication. Experiments intended to elucidate the mechanism(s) involved are also described.
It is now well-established that the replication of endogenous C-type RNA viruses in nonproducer avian and mammalian cells may be induced by exposure to certain chemical and physical agents, of which the halogenated pyrimidines are the most potent (Weiss et al., 1971; Rowe et al., 1971; Lowy et al., 1971; Aaronson, Todaro, and Scolnick, 1971). C57BL mouse embryo fibroblast (MEF) cells have been reported to carry the murine leukemia virus genome integrated in DNA form (Gelb et al., 1973). The radiation leukemia virus (RadLV) is a leukemogenie endogenous B-tropic virus which is extractable from thymic lymphomas induced by X irradiation of C57BL mice (Lieberman and Kaplan, 1959; Kaplan, 1967). Recently, activation of an endogenous, B-tropic, C-type virus from an established cell line of C57BL MEF treated with 5- bromodeoxyuridine (BUdR) in vitro has been reported (Lieberman et al., 1973). Attempts to induce virus replication in
MATERIALS
Culture medium. Cultures were grown in Eagle’s minimal essential medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 units/ml), streptomycin (100 pg/ml), and polymixin (50 units/ml). Cells. Secondary mouse embryo fibroblast (BL-MEF) cultures were prepared from C57BWKa mice, as previously described (Decleve et al., 1970); BL-5, an established line of C57BL/Ka mouse embryo fibroblasts, has been previously de-
‘This investigation was supported by Grants CA 03352 and CA 10372 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. 158 Copyright@ 1975 All rights
by of reproduction
Academic Press,Inc. in any form reserved.
AND METHODS
NIWA,
DECLlhE
scribed, as has BL-5(RadL.V), a subline of BL-5 stably infected with RadLV (Lieberman et al., 1973); NIH/3T3 and normal rat kidney (NRK) cells, which were obtained from Dr. Stuart Aaronson, National Cancer Institute, Bethesda, Maryland, have been described (Aaronson and Stephenson, 1973; Due-Nguyen et al., 1966); rat XC cells (Klement et al., 1969) were kindly provided by Dr. A. J. Hackett, Naval Biomedical Research Laboratory, Oakland, California; CH(GLV) cells are an established cell line (C,H-1) of C&I-MEF stably infected with Gross-AKR MuLV (GLV) (Decleve et al., 1974). Virus preparations. RadLV serially passaged in vitro, and designated RadLV*, was harvested from the supernatant fluids of 3-day-old BL5(RadLV) cell cultures (Lieberman et al., 1973). GLV, serially passaged in oitro and designated GLV*, was harvested from the supernatant fluids of 3-day-old C&I(GLV) cell cultures (Declkve et al., 1974). Virus assays. XC assays were performed according to the X-ray modification previously described for adaptation of the direct XC plaque procedure to the assay of RadLV (Niwa et al., 1973). Immunofluorescence (IF) assays were carried out as previously described (Decleve et al., 1974). IUdR treatment. Plastic Petri dishes (60 mm) were seeded with 2 x lo6 BL-MEF or BL-5 cells. On the following day, the cells were treated with the indicated concentration of IUdR for 24 hr, after which the IUdR-containing medium was removed and the cells were washed and infected with virus, as previously described (Niwa et al., 1973; Decleve et al., 1974). Two days after infection, the cells were overlaid with 2 x lo6 MEF cells in order to compensate for the cell killing effect of IUdR, and the cultures were incubated for 2 more days. At that time, the cells were X-irradiated with 5000 R, and 1 x lo8 XC cells were seeded on the dishes. Five days later, the dishes were fixed, and the number of plaques scored. When an IF assay was performed, the secondary overlay and X irradiation were
AND
159
KAPLAN
omitted. The cells were simply trypsinized and processed for indirect immunofluorescence 4 days after viral infection. Whenever necessary, the cell cultures were handled under special illumination (“Bug Lite,” General Electric Company), which emits no wavelengths shorter than 500 nm, in order to avoid the photodynamic action of visible light on IUdR (Wacker, 1963). Virus irradiation. Virus was diluted in phosphate-buffered saline (PBS). Virus samples were placed in watch dishes and gently swirled at 4” during UV irradiation with a germicidal lamp at 16.5 ergs/mm’/ sec. For X-ray irradiation, the virus samples were irradiated in plastic dishes with a 50 kVp twin beryllium window tube X-ray unit (Loevinger and Huisman, 1965) at a dose rate of 0.14 kradlsec. Cell irradiation. Twenty-four hours after cell seeding, medium was replaced with 2 ml of PBS. UV irradiation was performed at room temperature at a dose rate of 2.1 ergs/mm “/sec. RESULTS
Effect of IUdR on cell growth and plaque titer. BL-MEF cells were seeded onto
plates, together with varying concentrations of IUdR. After 24 hr, the IUdR-containing medium was removed, and the cells were rinsed with fresh medium. Cell growth was monitored daily thereafter. The doubling time of control cells under these conditions was approximately 24 hr in all experiments. IUdR has a marked depressing effect on the growth of BL-MEF cells (Fig. 1). It has been reported that RadLV replicates better in cells in log phase growth than in those in stationary phase (Niwa et al., 1973; Decleve et al., 1974). Therefore, the cell growth inhibitory effect of IUdR would have been expected to decrease the number of plaques observed. This expectation was not confirmed in an experiment in which cells were treated with 4 pg/ml of IUdR for 24 hr 1 day after seeding in order to avoid the lag phase. Then serial dilutions of a RadLV* preparation were assayed. Although titration patterns of the
160
EFFECT
OF
IODODEOXYURIDINE
IN
virus were of the one-hit type in both IUdR-treated and control cells, the virus titer was approximately twofold greater in the IUdR-treated cells (Fig. 2). Influence of IUdR concentration on virus titer. C57BL MEF cells were treated with various concentrations of IUdR, and virus dilutions (lo-’ to 10e5) were assayed by the XC test. IUdR alone at concentrations up to 100 pg/ml did not induce detectable amounts of endogenous virus from BLMEF cells in this short-term assay (Fig. 3). However, pretreatment of cells with IUdR markedly increased the number of plaque-
MOUSE
a d@z
EMBRYO
$-o-011•-0110-d
-
4t 0
L, 0
.,
1 1
FIBROBLASTS
1
10
IUdr OR Tdr CONCENTRATION bra/ml) I I ’ 1 10 100 lUdr CONCENTRATION (uM) I 10 100 Tdr CONCENTRATION WI
100
J I
FIG. 3. Influence of IUdR and TdR concentration on virus titer as detected by plaque assay. O---O = IUdR alone; B---W = TdR + virus; 04, 04, 04 = replicate experiments with IUdR + virus. RadLV* was used at concentrations of lo-’ and 10e5.
0
1
2
3
4
5
6
7
DAYS
FIG. 1. The effect of various doses of IUdR on C57BL MEF cell growth. 0 = 0 pg; 0 = 1 pg; n = 2 pg; 0 = 5 pg; A = 10 pg; A = 2 ag; 0 = 50 Pg.
10-3
10-5
10-4 VIRUS
DILUTION
FIG. 2. Titration curve of RadLV* on IUdR-treated cells (O--O) and control cells (04).
forming cells observed after infection with exogenous RadLV. The optimal concentration of IUdR for enhancing plaque yield was lo-40 PLM (4-10 pg/ml). In this concentration range, virus titer was increased nearly threefold, as compared with the control value. Higher doses of IUdR were less effective, possibly because the toxicity of IUdR overrides the enhancing effect. Since IUdR alone did not yield any plaques under these experimental conditions, the higher number of plaques found in cultures pretreated with IUdR cannot be ascribed to simple additive effects of exogenous virus and infectious endogenous virus induced by IUdR. Instead, the effect of IUdR on virus titer apparently involves the potentiation of cell response to infection by exogenous virus. Augmentation of infectivity of exogenous virus by IUdR pretreatment of indicator cells was also observed using the IF assay (data not shown). However, the increase in titer in this experiment was somewhat higher than that observed by the XC test. The potentiating effect is again observed at all virus concentrations by IF, and the titration curves are of the one-hit type for both the IUdR-treated cultures and the controls. Although the proportion of cells infected
NIWA.
DECLIhE
by exogenous RadLV was increased by IUdR pretreatment, indicating an increased susceptibility of the treated cells to infection, the yield of progeny virus per infected cell, as measured by titration of the supernatant fluids, was paradoxically usually somewhat decreased relative to that of control cultures (data not shown). This reduced yield of infectious virus was probably a secondary consequence of the inhibitory influence of IUdR on cell growth (Fig. l), since it has been shown that RadLV replication is closely coupled to cell replication (Declbve et al., 1975). Lack of effect of X-ray irradiation and secondary overlay on the potentiating effect of IUdR. X-ray irradiation of RadLV*-
infected test cells is a routine step in the modified XC assay used in this laboratory; it helps visualize plaques more clearly without decreasing the sensitivity of the assay, as does UV irradiation (Niwa et al., 1973). Since the halogenated pyrimidines are known to be effective radiosensitizers (Djordjevic and Szybalski, 1960; Kaplan, Smith, and Tomlin, 1962), it seemed important to determine whether the potentiating effect observed after IUdR treatment might be due to the synergism of IUdR and X irradiation. The data of Table 1 indicate that an X-ray dose of 5000 R did not influence the titer of exogenous virus in cells pretreated with IUdR. Moreover, the potentiating effect of IUdR was also obTABLE
1
LACK OF EFFECT OF X-RAY ON THE POTENTIATING ACTION OF IUdRa IUdR (rd
No. plaques
(5Z&
0
+ -
0.25
+ -
1
+ -
4
+ -
16
+ -
64
+ -
n RadLV*
was used at a lo-’
16 14 26 26 33 30 42 44 35 31 33 32 dilution.
AND
KAPLAN
161
served with the IF assay, which does not include an irradiation step. In all of the experiments reported here, a secondary overlay with BL-MEF cells was routinely used to compensate for the cell killing effect of IUdR (Fig. 1). The use of a secondary overlay has been reported to improve the sensitivity of the UV-XC cell test (Lowy et al., 1971; Teich et al., 1973). Several replicate experiments comparing plaque production with and without the use of a secondary overlay revealed that the overlay technique did not show any selectivity for IUdR-damaged cultures. Moreover, potentiation by IUdR was also seen with the IF assay, which does not involve a secondary overlay step. It is therefore concluded that neither X irradiation nor the secondary overlay procedure contributed to the potentiation response. Competitive inhibition by thymidine of the potentiating effect of IUdR. It has been
shown that the incorporation of IUdR into host-cell DNA is necessary for IUdR activation of murine leukemia virus (Teich et al., 1973). We therefore tested whether analog incorporation into host-cell DNA is also required for the potentiating action of IUdR on cell susceptibility to exogenous virus. Addition of an equal concentration of thymidine (TdR), a natural precursor of DNA synthesis which is a known competitive inhibitor of IUdR incorporation into DNA, completely blocks the potentiating effect of IUdR on relative plaque yield (Table 2). An equimolar concentration of TdR alone had no effect on exogenous virus titer (Fig. 3). Target size analysis of RadLV* on the IUdR pretreated cells. One possible mech-
anism of potentiation by IUdR postulates the complementation of defective exogenous virus particles by endogenous virus partially activated by IUdR. If this were so, the amount of genetic information required by exogenous virus in IUdR-treated cells should be less than that required in control cells. Inactivation of RadLV* by UV light was therefore studied to determine whether differences in viral genome target size are observed concomitantly with the potentiating effect of IUdR. The virus was irradiated with graded doses of UV light and
162
EFFECT
OF
IODODEOXYURIDINE
IN
assayed on control cells or on IUdR-(5 pg/ml) pretreated cells. Both inactivation curves were essentially exponential (with slight shoulders) and virtually identical (Fig. 4). The dose (D,,) required to inactivate RadLV to 37% of the initial titer was 745 ergs/mm’, which is comparable with values obtained for other MuLV (Kelloff et al., 1970; Nomura et al., 1972). Thus, the amount of viral genetic information required for RadLV* replication was not significantly less in IUdR-pretreated than in control cells. The same conclusion was reached from the study of X-ray survival curves of the virus on IUdR-treated and control cells (data not shown). Host range analysis of the progeny virus IUdR pretreatment of cells. If IUdR
after
potentiation
were the result of recombinaTABLE
COMPETITIVE
INHIBITION
2
OF IUdR
POTENTIATION
BY THYMIDINE
IUdR
TdR
0
0 0 6&ml
6rg/ml 6 &ml
100
I
Relative plaque number 100
170 loo
I
I
-
x
10-l
-
h
MOUSE
EMBRYO
FIBROBLASTS
tion of defective exogenous virus particles with partially activated endogenous virus, the hybrid viral progeny might be expected to differ from the input virus with respect to such attributes as host range. Accordingly, progeny virus from IUdR-pretreated, virus-infected cells was studied for its host range pattern. BL-5 cells are known to harbor B-tropic virus (Lieberman et al., 1973). Control and IUdR-pretreated BL-5 cells were infected with either RadLV* or GLV*. The tissue culture fluids taken 4 days after IUdR treatment were titered on BL-5, NIH-3T3, and NRK cells. IUdR pretreatment did not change the host range of RadLV* or GLV*, despite the fact that the latter was passaged once on BL-5 (Fv-lb) cells (Table 3). Potentiating effect of IUdR on exogenous virus replication in nonpermissive cells and in rat cells. It has been reported that GLV*
(an N-tropic virus), when titered on BLMEF (Fv-lb) cells, shows a two-hit titration pattern (Decleve et al., 1975). It seemed of interest to determine whether IUdR pretreatment of nonpermissive cells influences viral kinetics as well as susceptibility to infection. IUdR pretreatment of BL-MEF cells increased their sensitivity to GLV* by twofold, but the two-hit titration pattern of the virus remained unchanged (data not shown). Even though their efficiency of infection is low, both N-tropic and B-tropic MuLV can replicate on rat (NRK) cells (Decleve et al., 1975). An experiment was performed to test whether IUdR pretreatment (at 5 pg/ml) can also potentiate MuLV infection of NRK cells. As shown in Fig. 5, IUdR increased the plaque titer of GLV* on NRK cells by about twofold. Lack of potentiating irradiation. UV irradiation
10‘3
-
10-4
. 0
I 100 IRRADIATION
FIG.
measured (@A)
4. UV inactivation on IUdR-pretreated C57BL MEF.
I
I
200
300
TIME
curves (O--O)
(SEC)
of RadLV* as and control
effect
of
UV
of cells was studied for its possible potentiating effect on virus replication. BL-MEF cells were irradiated with UV (2.1 ergs/mm*/sec) at 24 hr after seeding and immediately thereafter infected with RadLV. Four days after infection, no potentiating effect was found by the XC assay (Fig. 6). Instead, there was a sigmoid inactivation of the susceptibility of cells to virus replication as a function of UV dose.
NIWA,
DECLEVE TABLE
INFLUENCE
OF IUdR
AND
163
KAPLAN
3
PRETREATMENT ON HOST-RANGE OF PROGENY VIRUS IN SUPEHNATANTS CULTURES INFECTED WITH RadLV* OR GLV*a Source of supernatant
Plaque
Supernatant dilution
no. on various
BL-5 No IUdR
RadLV* BL-5
infected cultures
GLV’ infected BL-5 cultures
IUdR
pretreated
RadLV* BL-5
infected cultures
GLV* infected BL-5 cultures
0 NOTE: from mice homozygous
TMTC TMTC 219 63 1 0
l/5 l/50 l/500 l/5 l/50 l/500
TMTC TMTC 37 57 2 0
NIH
CELL
test cells
3T3
NRK
0 0 0
0 0 0 0 0 0
TMTC TMTC 216 0 0 0
0 0 0 0 0 0
TMTC TMTC 248
RadLV* is a B-tropic murine leukemia virus, whereas GLV* is N-tropic. BL-5 cells are derived of strain C57BL, homozygous for Fv-I*; NIH 3T3 cells are derived from NIH Swiss mice, which are for Fv-1”; NRK cells are rat cells used to detect the possible induction of xenotropic virus.
11
10-z I 0 4-l
4-2 VIRUS
FIG. treated cells.
l/5 l/50 l/500 115 l/50 l/500
OF BL-5
5. Titration (O-48
4-3
DILUTIONS
curves of GLV* and control (O--O)
on IUdR-prerat (NRK)
DISCUSSION
These experiments have revealed a twoto threefold increase in cellular susceptibility to MuLV infection, as measured by IF-positive or plaque-forming cell number, in IUdR-pretreated cells infected by RadLV* or GLV*, without a concomitant change in viral replication kinetics or in the yield of infectious virus. Two possible explanations for the potentiating action of the halogenated pyrimidines, which may
10
20
IRRADIATION
FIG. 6. Effect of UV irradiation ity to virus infection.
30
40
TIME (SEC)
on cell susceptibil-
also be relevant to the enhancement of XC cell DNA transfection in BUdR-pretreated chicken fibroblasts (Svoboda et al., 19’73), have been considered: (1) induction of endogenous virus by the halogenated pyrimidine followed by complementation or recombination between endogenous and defective exogenous virus; and (21 enhancement of exogenous virus replication as a result of IUdR-induced DNA strand breakage, repair, and recombinational integration of the viral genome into cellular DNA.
164
EFFECT
OF IODODEOXYURIDINE
Animal virus preparations often contain defective particles carrying incomplete or damaged viral genomes. It is probable that our RadLV* and GLV* preparations similarly contain defective particles which cannot replicate unaided, even in permissive cells. If pretreatment of cells with IUdR partially activates an endogenous MuLV genome, the activated virus may rescue or may be rescued by the exogenous defective MuLV, resulting in an increased yield of virus of heterogeneous derivation. The interaction of exogenous Rous sarcoma virus with endogenous helper virus has been reported in chicken cells (Hanafusa et al., 1970; Hanafusa et al., 1972). However, several ts mutants of MuLV have been isolated which replicate stably at the permissive temperature without losing their ts character (Stephenson et al., 1972; Wong et al., 1973). One of these mutants, which also replicates poorly at the nonpermissive temperature, nevertheless yields progeny virus which retains its ts character. Thus, it would appear that recombination does not readily occur between endogenous and exogenous murine leukemia viruses. Evidence for the partial activation of C-type viruses also exists. Leukemia virus group-specific antigen appeared in BUdRtreated rat embryo cells in the absence of detectable infectious virus (Freeman et al., 1973). Group-specific antigens were also detected after short-term culture of thymocytes of various strains; again, infectious virus could not be detected (Lonai et al., 1974). Other studies on the spontaneous expression of C-type RNA virus genes detected by the appearance of group-specific antigen (Dougherty and Di Stefano, 1966; Heubner et al., 1970; Hanafusa et al., 1973; Parks et al., 1973) or of virus-specific RNA (Hayward and Hanafusa, 1973; Benveniste et al., 1973; Fan and Baltimore, 1973; Okabe et a1.,1973) in the absence of infectious virus also appear to involve partial activation. If the endogenous virus genome is partially activated by IUdR and requires complementation by defective exogenous virus for its complete expression, the amount of genetic information required for the exoge-
IN MOUSE
EMBRYO
FIBROBLASTS
nous virus to replicate should be less than that under normal conditions. This should be reflected in the relative target size of exogenous virus; the target size of the virus for UV irradiation should be smaller in the IUdR-pretreated cells than that in the control cells. A precedent exists in the case of bacteriophage in the well-known phenomenon of prophage reactivation (Jacob and Wollman, 1953; Blanc0 and Devoret, 1973). The relative target size of X phage is much smaller when the host cell is lysogenie for a homologous heteroimmune prophage. Prophage reactivation has also been observed with phage P, (Bertani and Bertani, 1971). The result shown in Fig. 4 seems to eliminate this possibility within the resolution of our methods. Host-range studies of progeny virus from IUdR-pretreated RadLV* - or GLV*-infected cells suggest that the progeny virus retained the original characteristics of the input exogenous virus. These experiments thus provide no evidence in support of the hypothesis that the progeny virus may represent recombinants of endogenous and exogenous virus (assuming that the assay methods used were sensitive enough to detect possible host-range variations). The alternative hypothesis involves the following considerations: type-C virus replication is now known to require the reverse transcription of viral RNA to the DNA form (Baltimore, 1970; Temin and Mizutani, 1970), followed by its integration into host-cell DNA (Balduzzi, 1973; Varmus et al., 1973; Markham and Baluda, 1973). The process of viral genome integration is thought to be through recombination between viral and host DNA, which requires the transient production of single-strand breaks in host DNA (Leis et al., 1973). IUdR is known to be a radiosensitizer for both UV and X-ray irradiation (Djordevic and Szybalski, 1960; Kaplan et al., 1962), creating single-strand breaks in cellular DNA where it replaces TdR (Wacker, 1963; Hotz and Walzer, 1970). It is therefore possible that IUdR enhances the integration of viral DNA into host-cell DNA by creating single-strand breaks in host-cell DNA, thus increasing the probability of
NIWA,
DECLEVE
viral DNA recombination with host-cell DNA. It is of interest that a two- to fourfold increase in the efficiency of transformation of mouse 3T3 cells by SV40, which also must integrate its genome into host-cell DNA, resulted when they were pretreated with IUdR or BUdR (Todaro and Green, 1964). Thus, although no direct evidence that IUdR enhances viral integration was provided by our experiments, the potentiating effect of halogenated pyrimidines on cellular susceptibility to infection by these viruses seems most plausibly attributed to such a mechanism. Moreover, whereas the photochemical reactions of halogenated pyrimidines in DNA lead to strand breaks, pyrimidine dimers are the major type of lesion in DNA damaged by UV irradiation. The fact that UV irradiation did not show any potentiating effect on cell susceptibility to MuLV infection suggests that the specific type of damage sustained by the host cell DNA may be an important determinant of the potentiation phenomenon. One-hit kinetics of UV inactivation of RadLV* is consistent with data reported for other C-type RNA viruses (Kelloff et al., 1970; Nomura et al., 1972; Friis, 1971; Levinson and Rubin, 1966; Rubin and Temin, 1959). The 60-70s RNA genome of C-type RNA viruses consists of 30-40s RNA subunits (Montagnier et al., 1969; Canaani et al., 1973). Biochemical studies of murine C-type virus replication suggest that these subunits are not redundant (Fan and Baltimore, 1973). The observed first order kinetics of virus inactivation by UV radiation would also be consistent with the absence of redundancy. REFERENCES AARONSON, S. A., and STEPHENSON, J. R. (1973). Independent segregation of loci for activation of biologically distinguishable RNA C-type viruses in mouse cells. Proc. Nut. Acad. Sci. USA 70, 2055-2058. AARONSON, S. A., TODARO, G. J., and SCOLNICK, E. M. (1971). Induction of murine C-type viruses from clonal lines of virus-free BALB/3T3 cells. Science 174, 157-159. BALDUZZI, P. C. (1973). Mechanism of oncogenic transformation by Rous sarcoma virus. III. Role of
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