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Studies on the effect of 5-fluoro-2′-deoxyuridine on polyoma T formation in mouse embryo cells
VIROLOGY
22, 368-376 (1964)
Studies
on the Effect of 5Fluoro-2’-Deoxyuridine T Formation
in Mouse
Embryo
on Polyoma Cells
ROSE SHEININ Subdivision
of Microbiology,
Division of Biological Research, The Ontario Institute, Toronto, Canada Accepted
November
Cancer
29,196s
Experiments in which FUdR has been employed to inhibit DNA synthesis, indicate that viral DNA is made no earlier than 30 minutes before the virion is formed in mouse embryo cells infected with polyoma virus. Viral protein was found to be synthesized under conditions in which replication of polyoma DNA was prevented. INTRODUCTION
Considerable evidence has been accumulated indicating that protein synthesis need not be obligatorily linked to DNA1 synthesis (Spiegelman, 1957). That specific viral protein formation can proceed in the absence of replication of specific viral DNA was suggested by the work of Reissig and Kaplan (1962). They observed that pseudorabies-like particles were formed in virusinfected cells incubated in the presence of fluorouracil, which prevented the synthesis of infectious virus. Recently, Volkin and Ruffilli (1962) demonstrated that proteins of bacteriophage T2 are made under conditions in which DNA synthesis is inhibited by FUdR. Similar reports of viral protein formation in the absence of vaccinia DNA synthesis have been made by Salzman et al. (1963) and by Shatkin (1963). The present communication deals with the synthesis of polyoma viral protein in the absence of DNA formation. This investigation arose in the course of studies of the multiplication of polyoma, a DNA-containing virus (DiMayorca et al., 1959; Smith et al., 1960), in mouse embryo cells. These experiments employed FUdR 1Abbreviations : DNA, deoxyribonucleic acid ; FUdR, 5-fluoro-2’-deoxyuridine ; PBS, phosphatebuffered saline, pH 7.2 ; PFU, plaque-forming units ; SBP, serum-blocking power.
as an inhibitor of DNA synthesis (Heidelberger, 1961; Cohen et al., 1958) to block the formation of infectious virus. Using this compound it was found that polyoma virus DNA is made just prior to the formation of the infectious particle. MATERIALS
Cultivation
AND METHODS
of mouse embryo cells and of
polyoma T. Procedures for the cultivation of mouse embryo cells and of stocks of polyoma T virus have been described (Sheinin, 1961, 1962). Measurement of polyoma T. The plaque assay for virus infectivity on mouse embryo cells, and the haemagglutination titration, using guinea pig red blood cells, have been described (Sheinin, 1961, I962). Experimental procedure for the study of polyoma T. formation in the presence of FUdR. Secondary cultures of mouse embryo
cells (in Brockway bottles) which were just approaching confluency were used throughout. For infection, the growth medium was decanted and the cells were washed 3 times with PBS (Dulbecco and Vogt, 1954). Virus (0.1 ml containing lo7 to log PFU/ml) was added, and the cultures were incubated at 37” for 90 minutes. After this interval, 5 ml of medium was added. The medium had the following basic composition; 1066 (Parker et al., 1957) minus thymidine minus
368
FUdR AND POLYOMA
coenzymes plus dialyzed foetal calf serum (5% v/v). For control cultures this medium was supplemented with thymidine at a final concentration of 4.1 x 1O-5 M; for test cultures it was supplemented with FUdR at a final concentration of 10-j M. The cultures were incubated at 37” in an incubator flushed with a gas mixture of 5% carbon dioxide in air and humidified to the extent of over 90%. The cultures were frozen and thawed 3 times prior to assay or subsequent processing. Experimental procedure for the study of the effect of FUdR on the incorporation of radioactive precursors into DNA and protein of mouse embryo cells. Secondary cultures as described above were employed. The medium was decanted, the cells were washed 3 times with PBS and were incubated with the desired medium. The media employed were the same as those described in the previous section, except that they contained the radioactive tracer noted in any given experiment under Results. To harvest, the radioactive medium was decanted and the cells were washed 3 times with medium 1066. The cells were removed from the glass by trypsinization using 1 ml of 0.1% Bactotrypsin in citrate-saline (Sheinin, 1961). The cells were harvested by centrifugation and frozen. The DNA or protein was isolated on Millipore filters by the method of Kahan (1960). The filters were transferred to planchettes, and the radioactivity was determined (on triplicate samples) using a D-47 gas-flow counter (Nuclear Chicago) with a 1 mg/cm” Mylar end window. Electron microscopy. Cells, scraped from the glass, were harvested by centrifugation and frozen at -20”. The cell pellets were processed in the frozen state and stained with phosphotungstic acid (Almeida and Howatson, 1963). In some instances, cells were embedded in methacrylate and thinsectioned. The preparations were examined in a Siemens electron microscope. Detection of polyoma antigen with jluorescent antibody. Antiserum to polyoma T virus, grown on mouse embryo cells, was prepared in New Zealand White rabbits. The serum was tested for the presence of
FORMATION
369
antibody by inhibition of plaque formation and by haemagglutination inhibition. Positive sera were treated with fluorescein isothyocyanate (British Drug Houses), and the fluorescein-coupled antibody was purified by the procedure of Coons (1956). Secondary cultures of mouse embryo cells were grown on glass slides in petri dishes. When the cells on the slides were contiguous, the medium was removed and 0.1 ml of virus was added to the centre of each slide, to infect the cultures as described above, To harvest, the slides were removed, processed, and examined with an ultraviolet microscope as described by Williams and Sheinin (1961). Total cell counts and counts of cells with nuclei exhibiting fluorescence were made on randomly selected areas. A minimum of 1000 cells was counted per slide. Duplicate slides were examined for each experimental count. Measurement of serum-blocking power. Noninfectious viral protein was measured by its ability to inhibit the action of antiserum to polyoma virus. This test for serumblocking power (SBP) of virus antigen was first worked out by De Mars (1955) for bacteriophage. To assess the amount of polyoma antigen formed in the presence of FUdR, the SBP of infected cultures grown in the presence of the inhibitor was compared wit,h that of infected noninhibited cultures inactivated by ultraviolet light. To 0.1 ml of a standard polyoma T antiserum was added 0.1 ml of the serum-blocking preparation. The volume was made up to 1.0 ml with PBS plus 1% (v/v) of foetal calf serum. The suspension was incubated at 37” for 2 hours. (Such incubation had been found to produce maximum inactivation by the antiserum, of plaque-formation by polyoma preparations.) Then, 0.1 ml of a standard polyoma preparation was added, and the suspension was again incubated at 37” for 2 hours. At this time dilutions were made into warm PBS plus 1% foetal calf serum and the samples were assayed for residual plaque-forming ability. Chemicals. FUdR was very kindly supplied by Dr. R.M. Fyfe of the Hoffman-LaRoche Company. Cytidine-2-C14 and val-
370
SHEININ
ine-2-Cl4 were obtained from the Schwarz Biochemical Company. RESULTS
Effect of FUdR on the Synthesis of DNA in Mouse Embryo Cells Several investigators have reported that FUdR inhibits the synthesis of DNA in
bacteria and in animal cells (Heidelberger, 1961; Cohen et al., 1958). Before proceeding to studies of the effect of this compound on polyoma virus formation, it was necessary to demonstrate a similar effect in mouse embryo cells. As is shown in Fig. 1, FUdR was found to inhibit totally and immediately the incorporation of cytidine-2-W4 into the DNA of these cells. The inhibition was reversed by thymidine. Cultures which had been incubated for as long as 18 hours in FUdR, when subsequently incubated in its absence, incorporated cytidine-2-Cl4 into DNA at approximately the same rate as did uninhibited cultures. Effect of FUdR on Protein Mouse Embryo Cells
$ ‘0 5 P s t
--,-0
,‘A----e-i-
IO 20 30 40 50 Period of lncubotion at 37°C (hours)
FIG. 1. Effect of FUdR on the incorporation of of mouse embryo cytidine-2-C I4 into the DNA cells. Secondary cultures of mouse embryo cells (approximately 4 X 10’ cells) were incubated, as described under Materials and Methods, in media containing and lacking FUdR and supplemented
with cytidine-2-P
(0.05 PC/ml). After various
intervals the DNA was isolated from the cells and its radioactivity determined. Control cultures, a-0 ; cultures containing FUdR, X-X. To study the reversibility of FUdR inhibition by thymidine, cultures were incubated as above with FUdR for 10 (O-O) and 18 (0-O) hours, respectively. The medium was decanted, the cell layers were. washed 3 times with 1066, and 5 ml of control medium was added. Cultures were incubated for the further intervals plotted; the DNA was extracted and its radioactivity measured. Each point is the average value obtained from triplicate cultures.
Synthesis
in
Since FUdR is known to inhibit specifically the synthesis of DNA, it would be expected that all protein synthesis except that dependent on de novo DNA formation, should continue in the presence of the inhibitor. This expectation was fulfilled in experiments like that shown in Fig. 2. FUdR did not inhibit the incorporation of valine2-C14 into the proteins of mouse embryo cells. Indeed, whereas protein synthesis came to a halt in control cultures when cell division ceased (after approximately 24 hours in the experiment depicted), incorporation of amino acid into protein in inhibited cultures continued at an undiminished rate for between 36 and 40 hours. Effect of FUdR on the Formation fectious Polyoma T
of In-
Having established that FUdR totally blocks DNA formation in mouse embryo cells, the effect of this compound on polyoma T formation was examined. Infected mouse embryo cultures were incubated with FUdR, at various concentrations, for 24 hours, and the amount of infectious virus formed was determined by plaque assay. It was found that although variable results were obtained with FUdR at concentrations of 10e6 M and lo-? M, complete inhibition’was always achieved at 10W5M. Therefore, this concentration was employed in all subsequent investigations. To examine the effect of this compound during the l&hour latent period, cultures
FUdR AND POLYOMA
371
FORMATION
or the total yield obtained. If the analogue was left in the cultures for 18 hours, reversal with thymidine was followed by a lag in polyoma synthesis, although the rate of virus synthesis when it resumed was essentially the same as that in uninhibited cultures. Incubation with FUdR for periods beyond 18 hours resulted in an increased lag prior to onset of virus formation and a decreased rate of synthesis.
18,000-
d
0
20 30 40 IO Period of Incubation ot 37’C
50 (hours)
FIG. 2. Effect of FUdR on the incorporation of valine-2-C” into the protein of mouse embryo cells. Secondary mouse embryo cells were incubated, as described under Materials and Methods, in media containing or lacking FUdR, and supplemented with valine-2-C” (0.37 &ml). Cultures were harvested as described, the protein was isolated and its radioactivity determined. Control cultures, 0-O ; cultures containing FUdR, 0-O.
were incubated for varying periods with FUdR (see Materials and Methods).. The FUdR-containing medium was then removed and replaced with thymidine-containing medium. The cells were reincubated, samples were harvested at/ subsequent intervals, and their polyoma content was determined. It was found that the inhibitory effect of FUdR was reversible throughout almost the entire latent period. In Fig. 3 are shown curves for control cultures and cultures incubated with FUdR for 17l/s hours and for 18 hours. Curves similar to those obtained with control cultures and those treated for 17% hours have been obtained for cultures incubated with inhibitor for any interval up to 17iY.z hours. It was found that if the analogue was removed up to 17i/s hours after infection, there was no detectable effect on the time of onset of virus formation, the rate of viral synthesis,
FIG. 3. Effect of FUdR on the synthesis of infectious polyoma T virus in mouse embryo cells. Secondary mouse embryo cultures were infected with polyoma T and incubated in the presence and absence of FUdR: control cultures, O-O ; plus FUdR, x-x. To study reversibility of the action of FUdR, FUdR-containing medium was decanted at 17% (V----V), and 18 (O-- -0) hours, respectively. The cells were washed 3 times with PBS, and control medium was added. The cultures were reincubated at 37°C. Samples were removed at subsequent intervals, and the polyoma virus formed was determined.
372
SHEININ
Formation of Polyoma Antigen in the Presence of FUdR The above experiments indicate that FUdR, which prevented DNA synthesis in mouse embryo cells, also inhibited the formation of infectious polyoma virus. It was of interest to determine whether, under these circumstances, polyoma protein was made. Detection of specific polyoma protein formation was carried out using the technique of fluorescent antibody staining (Coons, 1956). Slide cultures were infected with polyoma T virus (see Materials and Methods) and incubated for 2448 hours in the presence or absence of FUdR. The slides were removed and processed, and the cells were stained with fluorescein-labelled antipolyoma y-globulin isolated from immune rabbit serum. Counts were made of total cells and cells with nuclei exhibiting fluorescence. Under the conditions employed at present, only a small proportion (l-10%) of mouse embryo cells treated with polyoma virus, even at input multiplicities of 1000, are infected, if virus production is the criterion of infectivity. This difficulty has been encountered by other workers (Winocour and Sachs, 1960). However, it is clear from the data presented in Table 1 that essentially the same number of cells with fluorescing nuclei appeared in cultures incubated with and without FUdR, although no infectious virus was formed in the presence of the analoguc. It may be concluded, therefore, that polyoma antigen is synthesized under conditions in which no DNA and no infectious virus are made.
Electron
Microscopic
Studies
It was reported by Reissig and Kaplan (1962) that when chick embryo cells infected with pseudorabies virus were incubated with fluorouracil, characteristic viruslike particles were formed. The observation made in the foregoing experiment that in the presence of FUdR, nuclei exhibiting fluorescence appeared in infected cultures suggested that polyoma-like particles might be formed in the presence of this inhibitor. lMouse embryo cultures were infected with polyoma T and incubated in the prcscnce and absence of FUdR. Examination of cell preparations with the electron microscope (see Materials and Methods) after periods of 2448 hours, revealed that particles morphologically characteristic of polyoma virus (Howatson and Almeida, 1960) were present in the nuclei of cells of control and inhibited cultures (Fig. 4). The nuclear membrane and cytoplasmic vesicles are seen on the right edge of Figs, 4A and 4B. The characteristic polyoma-like morphology of particles formed in the presence of FUdR is seen in Fig. 4C. Such observations have been made with freshly-frozen material and with preparations embedded in methacrylate prior to sectioning. Quantitative Particles FUdR
Estimate of Polyoma-like Formed in the Presence of
In addition to the qualitative evidence obtained above for the formation of polyoma protein in the absence of DNA synthesis, a quantitative estimate of such protein formation was sought. One approach to this
TABLE
1
DETECTION BY FLUORESCENT ANTIBODY STAINING OF MOUSE EMBRYO CELLS CVNTAINING POLYOMA ANTIGEN IN INFECTED CULTURES INCUBATED IN THE PRESENCE AND ABSENCE OF FUdR Expt. no. 1
2
Period of incubation (hr)
Per cent fluorescent nuclei
PFU/ml - FUdR
+FUdR
- FUdK
+FUdR
0
3.0 x 10”
-
-
24 36
3.9 x 106 3.9 x 106
8.2 X lo4 5.8 x 104
5.4 8.3
5.7 10.9
0 48
2.5 X lo4 2.9 x 106
9.5 x 103
5.3
4.0
FIG. 4. Electron micrographs of the nuclear region of mouse embryo cells infected with polyoma T virus in the presence and absence of FUdR. Preparations shown in (A) and (B) wvc:re embedded in methacrylate and thin-sectioned. The preparation shown in (C) was frozen at -20°C and sectioned just prior to staining in phosphotungstic acid. A. Control culture, incubated wkhout FUdR. Magnification: X 50,000. B. Culture incubated with FUdR. Magnification: X 50,000. C. Similar to (B). Magnification: X 240,000.
problem was to count the numbers of viruslike particles formed in the presence and absence of FUdR. Mouse embryo cultures were infected and incubated with or without analogue for 32 hours. The culture lysates were spun at 8000 9 for 30 minutes to sediment cell debris. The supernatants were then centrifuged at 40,000 9 for 1 hour. The resulting pellets wcrc resuspended in PBS, and the suspensions were spun again at 8000 and 40,000 g as above. The pellets were taken up in 2 ml PBS and subjected to sonic vibrations at 10,000 kilocycles per second for 2 minutes. A volume of the suspensions was mixed with a known concentration of latex part,icles. These mixtures were treated with phosphotungstic acid and examined with the electron microscope. Count’s were made of latex particles and pnrticlcs with characteristic polyoma morphology and size. From these count.s was calculated the number of such part,icles formed in cultures incubated in the presence and absence of FUdR (Table 2). Although the plaque-forming units prescnt in the control culture after 32 hours
TABLE
2
QUAP*TIT,\TIVE I~ETERMINATION OF POLYOM.\-LIKE IN INPS~TICLES FORTNED AFTER 32 HOURS FECTED MORSE EJIBRYO CELLS INCUB.YTED IN THE PRESENCE IND ABSENCE OF FUdR.
Particle counW
Test material
PFU/ml
Control
4.90 x 108 1.36 x 109 1.03 X 103
9.5 x 106
8.10 4.90 1.76 1.52 1.31 1.58
1.8
Plus FUdR
Zero-time
culture
x x x X X x
No part.icles tectable
10” 108 IOY 10y
x
103
109
109 de-
2.1 X 104
a Data presented here were obtained using different sanlples prepared from one experiment.
cxcceded those present in the inhibited cultllre by a factor of almost 101, the number of virus-like particles in both cultures was t,he sa111e.
374
SHEININ
Measurement of Polyoma Antigen Formed in the Presence of FUdR by SerumBlocking Power A second and more specific quantitative determination of the amount of polyoma protein formed in the presence of FUdR was made using serum-blocking power (SBP) as criterion. The use of SBP for the study of noninfectious viral protein was introduced by De Mars (1955). He demonstrated that protein of bacteriophage T2, present as noninfectious virus, can be accurately measured by its ability to compete with intact phage for phage-specific antibody. The SBP of infected cultures incubated for 36 hours in the presence of FUdR, was compared with that of control cultures in which infectious virus was formed. Portions of the control cultures, harvested after 36 hours and frozen and thawed 3 times, were subjected to ultraviolet light (2537 A, 5 ergs/ mm”/sec) for 2 hours at approximately 10”. Such treatment, results in the loss of the plaque-forming ability of polyoma virus (G. F. Whitmore, 1963, personal communication). In the experiments described herein infectivity of the virus lysates was reduced by at least a factor of 106. However, the TABLE
3
MEASUREMENT OF SBP IN POLYOMA-INFECTED MOUSE EMBRYO CULTURES INCUBATED IN THE PRESENCE AND ABSENCE OF FUdR
Relative concentration of preparation
1 50 loo 200 500 1000 0
SBP! measured as the fractional survival of test virus, incubated with a standard immune serum, of the following: c-UV”
Fb
0.009
0.006 0.006 0.04 0.02 0.02 1.0 0.008
0.01 0.02 0.04 1.0 0.008
a C-UV, Control culture was incubated in the absence of FUdR. It produced infectious virus. For SBP assay, it was inactivated with ultraviolet light. b F, Culture incubated in the presence of FUdR.
haemagglutinating capacity and the particle morphology of the virus preparations remained unaffected. Preparations treated with ultraviolet light exhibited SBP. A comparison of the SBP power of control cultures containing infectious virus which were first inactivated by ultraviolet light, with t,hat of cultures prepared in the presence of FUdR, is shown in Table 3. The data show that approximately the same amount of serum-blocking material was formed in the presence and absence of FUdR, suggesting that the absence of DNA synthesis in the inhibited cultures has not interfered with the formation of specific polyoma protein. DISCUSSION
The presence of FUdR in cultures of mouse embryo cells infected with polyoma virus for up to 17l/2 hours has no effect on the subsequent time course of virus formation when the inhibitor is removed. It is therefore likely that viral DNA is synthesized very late during the latent period, i.e., no more than 30 minutes prior to the formation of intact virions. This conclusion is supported by the finding of R. Weil (1962, personal communication) that infectious DNA cannot be isolated from polyomainfected cells at any time prior to that at which intact virus first begins to appear. It is also indicated by the observation (Williams and Sheinin, 1961), that viral DNA, detected by acridine orange staining and deoxyribonuclease resistance, cannot be observed in the nuclei of infected cells before 18-20 hours after infection. The fact that FUdR-inhibition can be reversed up to 17l% hours with no effect on subsequent virus formation suggests that all the processes involved in polyoma reproduction, except replication of viral DNA, can proceed in the absence of synthesis of DNA, and in particular of viral DNA. Whereas FUdR prevents the synthesis of polyoma DNA,2 it does not appear to interfere with the formation of specific viral protein. All the available evidence indicates ’ Preliminary experiments indicate that particles formed in polyoma-infected cells in the presence of FUdR contain little or no nucleic acid.
FUdR
AND
POLYOMA
that the amount of viral protein formed in inhibited cultures is the same as that made in uninhibit,ed cells. Thus it seems likely that the original genetic material which enters the cells is able to direct the formation of all the new virus protein molecules formed. The formation of specific viral protein in the presence of FUdR has been reported for a number of DNA-containing viruses (Volkin and Ruffilli, 1962; Tanami et al., 1961; Salzman et al., 1963; Shatkin, 1963). These findings indicate that the replication of virus DNA may not necessarily be correlated with the other functions of this DNA in infected cells. There is, to date, one exception to the above observations. It was reported by Flanagan and Ginsberg (1962) and by Kjellen (1962) that adenovirus protein is not formed in the presence of FUdR. The significance of this finding is not clear, but may be related to the formation of a nonviral saline-soluble DNA associated with adenovirus infection (Ginsberg and Dixon, 1961). The demonstration that in the absence of DNA formation both polyoma antigen and morphologically characteristic viruslike part,icles are produced, indicates that the polyoma protein formed must be essentially the same at the level of primary and tertiary structure as that in infectious virus. Casper and Klug (1962) suggested that viral morphology was largely a function of the protein and that once formed, this protein could take up its characteristic morphological structure without linkage to DNA. This prediction has been borne out by the demonstration that virus-like particles are formed in the absence of DNA formation as a result of infection by polyoma virus and of pseudorabies virus (Reissig and Kaplan, 1962). ACKNOWLEDGMENTS The author’s grateful thanks go to Mr. Jim Morrison. His careful and thoughtful technical assistance is an essential part of the work reported. The critical comments of Dr. Louis Siminovitch throughout the course of this work have been most welcome. Mrs. June D. Almeida cheerfully coped with the electron microscopic studies.
375
FORMATION
Mr. Robert Escoffery prepared the fluoresceincoupled polyoma antiserum and carried out the fluorescent antibody labeling. This work was aided financially by grants from the National Cancer Institute of Canada, and by the U.S. Department of Public Health, Grants #AI-04229(VR), and #CA-04964. REFERENCES ALMEIDA, J. D., and HOWATSON, A. F. (19631. A negative staining method for cell-associated virus. J. Cell Biol. 16, 61&620. CASPAR, D. L. D., and KLUG, A. (1962). Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27, l-24. COHEN, S. S., FLAKS, J. G., BARNER, H. D., LOEB, M. R., and LICHTENSTEIN, J. (1958). The mode of action of 5-fluorouracil and its derivatives. Proc. Natl. Acad. Sci. U. S. 44,1004-1012. COONS, A. H. (1956). Histochemistry with labelled antibody. Intern. Rev. Cytol. 5, 1-23. DE MARS, R. I. (1955). The production of phagerelated materials when bacteriophage development is interrupted by proflavine. Virology 1, 83-99. DIMAYORCA, G. A., EDDY, B. E., STEWART, S. E., HUSTER, W. S., FRIEND, C., and BENDICH, A. (1959). Isolation of infectious deoxyribonucleic acid from SE polyoma-infected tissue cultures. Proc. Natl. Acad. Sci. U. S. 45, 1805-1808. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Eqcptl. h4ed. 99, 167-182. FLANAGAN, J. F., and GINSBERG, H. S. (1962). Synthesis of virus-specific polymers in adenovirusinfected cells: effect of 5-fluorodeoxyuridine. J. Exptl. Med. 116, 141-157. GINSBERG, H. S., and DIXON, M. K. (1961). Nucleic acid synthesis in Types 4 and 5 Adenovirus-infected HeLa cells. J. Exptl. Med. 113, 283-299. HEIDELBERGER, C. ( 1961) Nucleic acid synthesis and mechanism of action of fluoropyrimidines. In “Biological Approaches to Cancer Chemotherapy” (R. J. C. Harris, ed.), pp. 47-58. Academic Press, New York. HOWATSON, A. F., and ALMEIDA, J. D. (1960). Observations on the fine structure of polyoma virus. J. Biophys. Biochem. Cytol. 8, 828-834. KAHAN, F. M. (1960). Purification and measurement of microgram amounts of radioactive nucleic acids and proteins from animal cells in tissue culture. Anal. Biochem. 1, 107-126. KJELL~N, L. (1962). Effect of 5-halogenated pyrimidines on cell proliferation and adenovirus multiplication. Virology 18, 6670.
376
SHEININ
PAHKEH, R. C., CASTOR, I,. N., and MCCULLOCH, E. A. (1957). Altered cell strains in continuous culture: A general survey. Spec. Publ. N. Y. Acad. Sci. 5, 303-313. REISSIG, M., and KAPLAN, A. S. (1962). The morphology of noninfective pseudorabies virus produced by cells treated with 5-fluorouracil. T/idogy 16, 1-8. SALZMAK,N. P., SHATKIN, A. J., and SEBHING, E. D. (1963). Viral protein and DNA synthesis in vaccinia virus-infected HeLa cell cultures. l’irology 19, 542-550. SHATKIN, A. J. (1963). The formation of vaccinia virus protein in the presence of 5-fluorodeoxyuridine. Virology 20,292-301. SMITH, J. D., FREEMAN, G., VOGT, M., and DULBECCO, R. (1960). The nucleic acid of polyoma virus. Virology 12, 185-196. SHEINIR., R. (1961). A rapid plaque assay for polyoma virus. Virology 15, 85-87.
SHEINIK, R. (1962). Procedures for the purification of polyoma T virus. Virology 17, 426440. SPIEGELMAN, S. (1957). Nucleic acids and the synthesis of proteins. Symp. Chem. Basis Heredity, Baltimore, 1958. Johns Hopkins Univ. McCollumPratt Inst. Contrib. 153, 232-267. TAKAMI, Y., POLLARD, M., and STARR, T. J. (1961). Replication pattern of psittacosis virus in a tissue culture system. Virology 15, 22-29. VOLKIN, E., and RUFFILLI, A. (1962). Dissociation of macromolecular synthetic processes in T2infected Escherichia coli. Proc. Natl. Acad. Sci. U. S. 48, 2193-2200. WILLIAMS, M. G., and SHEIKIN, R. (1961). Cytological skdies of mouse embryo cells infected with polyoma virus, using acridine orange and fluorescent antibody. Virology 13, 368370. WINOCOUR, E., and SACHS, L. (1960). Cell-virus interactions with polyoma virus. I. Studies on the lytic interaction in the mouse embryo system. Virology 11, 699-721.
22, 368-376 (1964)
Studies
on the Effect of 5Fluoro-2’-Deoxyuridine T Formation
in Mouse
Embryo
on Polyoma Cells
ROSE SHEININ Subdivision
of Microbiology,
Division of Biological Research, The Ontario Institute, Toronto, Canada Accepted
November
Cancer
29,196s
Experiments in which FUdR has been employed to inhibit DNA synthesis, indicate that viral DNA is made no earlier than 30 minutes before the virion is formed in mouse embryo cells infected with polyoma virus. Viral protein was found to be synthesized under conditions in which replication of polyoma DNA was prevented. INTRODUCTION
Considerable evidence has been accumulated indicating that protein synthesis need not be obligatorily linked to DNA1 synthesis (Spiegelman, 1957). That specific viral protein formation can proceed in the absence of replication of specific viral DNA was suggested by the work of Reissig and Kaplan (1962). They observed that pseudorabies-like particles were formed in virusinfected cells incubated in the presence of fluorouracil, which prevented the synthesis of infectious virus. Recently, Volkin and Ruffilli (1962) demonstrated that proteins of bacteriophage T2 are made under conditions in which DNA synthesis is inhibited by FUdR. Similar reports of viral protein formation in the absence of vaccinia DNA synthesis have been made by Salzman et al. (1963) and by Shatkin (1963). The present communication deals with the synthesis of polyoma viral protein in the absence of DNA formation. This investigation arose in the course of studies of the multiplication of polyoma, a DNA-containing virus (DiMayorca et al., 1959; Smith et al., 1960), in mouse embryo cells. These experiments employed FUdR 1Abbreviations : DNA, deoxyribonucleic acid ; FUdR, 5-fluoro-2’-deoxyuridine ; PBS, phosphatebuffered saline, pH 7.2 ; PFU, plaque-forming units ; SBP, serum-blocking power.
as an inhibitor of DNA synthesis (Heidelberger, 1961; Cohen et al., 1958) to block the formation of infectious virus. Using this compound it was found that polyoma virus DNA is made just prior to the formation of the infectious particle. MATERIALS
Cultivation
AND METHODS
of mouse embryo cells and of
polyoma T. Procedures for the cultivation of mouse embryo cells and of stocks of polyoma T virus have been described (Sheinin, 1961, 1962). Measurement of polyoma T. The plaque assay for virus infectivity on mouse embryo cells, and the haemagglutination titration, using guinea pig red blood cells, have been described (Sheinin, 1961, I962). Experimental procedure for the study of polyoma T. formation in the presence of FUdR. Secondary cultures of mouse embryo
cells (in Brockway bottles) which were just approaching confluency were used throughout. For infection, the growth medium was decanted and the cells were washed 3 times with PBS (Dulbecco and Vogt, 1954). Virus (0.1 ml containing lo7 to log PFU/ml) was added, and the cultures were incubated at 37” for 90 minutes. After this interval, 5 ml of medium was added. The medium had the following basic composition; 1066 (Parker et al., 1957) minus thymidine minus
368
FUdR AND POLYOMA
coenzymes plus dialyzed foetal calf serum (5% v/v). For control cultures this medium was supplemented with thymidine at a final concentration of 4.1 x 1O-5 M; for test cultures it was supplemented with FUdR at a final concentration of 10-j M. The cultures were incubated at 37” in an incubator flushed with a gas mixture of 5% carbon dioxide in air and humidified to the extent of over 90%. The cultures were frozen and thawed 3 times prior to assay or subsequent processing. Experimental procedure for the study of the effect of FUdR on the incorporation of radioactive precursors into DNA and protein of mouse embryo cells. Secondary cultures as described above were employed. The medium was decanted, the cells were washed 3 times with PBS and were incubated with the desired medium. The media employed were the same as those described in the previous section, except that they contained the radioactive tracer noted in any given experiment under Results. To harvest, the radioactive medium was decanted and the cells were washed 3 times with medium 1066. The cells were removed from the glass by trypsinization using 1 ml of 0.1% Bactotrypsin in citrate-saline (Sheinin, 1961). The cells were harvested by centrifugation and frozen. The DNA or protein was isolated on Millipore filters by the method of Kahan (1960). The filters were transferred to planchettes, and the radioactivity was determined (on triplicate samples) using a D-47 gas-flow counter (Nuclear Chicago) with a 1 mg/cm” Mylar end window. Electron microscopy. Cells, scraped from the glass, were harvested by centrifugation and frozen at -20”. The cell pellets were processed in the frozen state and stained with phosphotungstic acid (Almeida and Howatson, 1963). In some instances, cells were embedded in methacrylate and thinsectioned. The preparations were examined in a Siemens electron microscope. Detection of polyoma antigen with jluorescent antibody. Antiserum to polyoma T virus, grown on mouse embryo cells, was prepared in New Zealand White rabbits. The serum was tested for the presence of
FORMATION
369
antibody by inhibition of plaque formation and by haemagglutination inhibition. Positive sera were treated with fluorescein isothyocyanate (British Drug Houses), and the fluorescein-coupled antibody was purified by the procedure of Coons (1956). Secondary cultures of mouse embryo cells were grown on glass slides in petri dishes. When the cells on the slides were contiguous, the medium was removed and 0.1 ml of virus was added to the centre of each slide, to infect the cultures as described above, To harvest, the slides were removed, processed, and examined with an ultraviolet microscope as described by Williams and Sheinin (1961). Total cell counts and counts of cells with nuclei exhibiting fluorescence were made on randomly selected areas. A minimum of 1000 cells was counted per slide. Duplicate slides were examined for each experimental count. Measurement of serum-blocking power. Noninfectious viral protein was measured by its ability to inhibit the action of antiserum to polyoma virus. This test for serumblocking power (SBP) of virus antigen was first worked out by De Mars (1955) for bacteriophage. To assess the amount of polyoma antigen formed in the presence of FUdR, the SBP of infected cultures grown in the presence of the inhibitor was compared wit,h that of infected noninhibited cultures inactivated by ultraviolet light. To 0.1 ml of a standard polyoma T antiserum was added 0.1 ml of the serum-blocking preparation. The volume was made up to 1.0 ml with PBS plus 1% (v/v) of foetal calf serum. The suspension was incubated at 37” for 2 hours. (Such incubation had been found to produce maximum inactivation by the antiserum, of plaque-formation by polyoma preparations.) Then, 0.1 ml of a standard polyoma preparation was added, and the suspension was again incubated at 37” for 2 hours. At this time dilutions were made into warm PBS plus 1% foetal calf serum and the samples were assayed for residual plaque-forming ability. Chemicals. FUdR was very kindly supplied by Dr. R.M. Fyfe of the Hoffman-LaRoche Company. Cytidine-2-C14 and val-
370
SHEININ
ine-2-Cl4 were obtained from the Schwarz Biochemical Company. RESULTS
Effect of FUdR on the Synthesis of DNA in Mouse Embryo Cells Several investigators have reported that FUdR inhibits the synthesis of DNA in
bacteria and in animal cells (Heidelberger, 1961; Cohen et al., 1958). Before proceeding to studies of the effect of this compound on polyoma virus formation, it was necessary to demonstrate a similar effect in mouse embryo cells. As is shown in Fig. 1, FUdR was found to inhibit totally and immediately the incorporation of cytidine-2-W4 into the DNA of these cells. The inhibition was reversed by thymidine. Cultures which had been incubated for as long as 18 hours in FUdR, when subsequently incubated in its absence, incorporated cytidine-2-Cl4 into DNA at approximately the same rate as did uninhibited cultures. Effect of FUdR on Protein Mouse Embryo Cells
$ ‘0 5 P s t
--,-0
,‘A----e-i-
IO 20 30 40 50 Period of lncubotion at 37°C (hours)
FIG. 1. Effect of FUdR on the incorporation of of mouse embryo cytidine-2-C I4 into the DNA cells. Secondary cultures of mouse embryo cells (approximately 4 X 10’ cells) were incubated, as described under Materials and Methods, in media containing and lacking FUdR and supplemented
with cytidine-2-P
(0.05 PC/ml). After various
intervals the DNA was isolated from the cells and its radioactivity determined. Control cultures, a-0 ; cultures containing FUdR, X-X. To study the reversibility of FUdR inhibition by thymidine, cultures were incubated as above with FUdR for 10 (O-O) and 18 (0-O) hours, respectively. The medium was decanted, the cell layers were. washed 3 times with 1066, and 5 ml of control medium was added. Cultures were incubated for the further intervals plotted; the DNA was extracted and its radioactivity measured. Each point is the average value obtained from triplicate cultures.
Synthesis
in
Since FUdR is known to inhibit specifically the synthesis of DNA, it would be expected that all protein synthesis except that dependent on de novo DNA formation, should continue in the presence of the inhibitor. This expectation was fulfilled in experiments like that shown in Fig. 2. FUdR did not inhibit the incorporation of valine2-C14 into the proteins of mouse embryo cells. Indeed, whereas protein synthesis came to a halt in control cultures when cell division ceased (after approximately 24 hours in the experiment depicted), incorporation of amino acid into protein in inhibited cultures continued at an undiminished rate for between 36 and 40 hours. Effect of FUdR on the Formation fectious Polyoma T
of In-
Having established that FUdR totally blocks DNA formation in mouse embryo cells, the effect of this compound on polyoma T formation was examined. Infected mouse embryo cultures were incubated with FUdR, at various concentrations, for 24 hours, and the amount of infectious virus formed was determined by plaque assay. It was found that although variable results were obtained with FUdR at concentrations of 10e6 M and lo-? M, complete inhibition’was always achieved at 10W5M. Therefore, this concentration was employed in all subsequent investigations. To examine the effect of this compound during the l&hour latent period, cultures
FUdR AND POLYOMA
371
FORMATION
or the total yield obtained. If the analogue was left in the cultures for 18 hours, reversal with thymidine was followed by a lag in polyoma synthesis, although the rate of virus synthesis when it resumed was essentially the same as that in uninhibited cultures. Incubation with FUdR for periods beyond 18 hours resulted in an increased lag prior to onset of virus formation and a decreased rate of synthesis.
18,000-
d
0
20 30 40 IO Period of Incubation ot 37’C
50 (hours)
FIG. 2. Effect of FUdR on the incorporation of valine-2-C” into the protein of mouse embryo cells. Secondary mouse embryo cells were incubated, as described under Materials and Methods, in media containing or lacking FUdR, and supplemented with valine-2-C” (0.37 &ml). Cultures were harvested as described, the protein was isolated and its radioactivity determined. Control cultures, 0-O ; cultures containing FUdR, 0-O.
were incubated for varying periods with FUdR (see Materials and Methods).. The FUdR-containing medium was then removed and replaced with thymidine-containing medium. The cells were reincubated, samples were harvested at/ subsequent intervals, and their polyoma content was determined. It was found that the inhibitory effect of FUdR was reversible throughout almost the entire latent period. In Fig. 3 are shown curves for control cultures and cultures incubated with FUdR for 17l/s hours and for 18 hours. Curves similar to those obtained with control cultures and those treated for 17% hours have been obtained for cultures incubated with inhibitor for any interval up to 17iY.z hours. It was found that if the analogue was removed up to 17i/s hours after infection, there was no detectable effect on the time of onset of virus formation, the rate of viral synthesis,
FIG. 3. Effect of FUdR on the synthesis of infectious polyoma T virus in mouse embryo cells. Secondary mouse embryo cultures were infected with polyoma T and incubated in the presence and absence of FUdR: control cultures, O-O ; plus FUdR, x-x. To study reversibility of the action of FUdR, FUdR-containing medium was decanted at 17% (V----V), and 18 (O-- -0) hours, respectively. The cells were washed 3 times with PBS, and control medium was added. The cultures were reincubated at 37°C. Samples were removed at subsequent intervals, and the polyoma virus formed was determined.
372
SHEININ
Formation of Polyoma Antigen in the Presence of FUdR The above experiments indicate that FUdR, which prevented DNA synthesis in mouse embryo cells, also inhibited the formation of infectious polyoma virus. It was of interest to determine whether, under these circumstances, polyoma protein was made. Detection of specific polyoma protein formation was carried out using the technique of fluorescent antibody staining (Coons, 1956). Slide cultures were infected with polyoma T virus (see Materials and Methods) and incubated for 2448 hours in the presence or absence of FUdR. The slides were removed and processed, and the cells were stained with fluorescein-labelled antipolyoma y-globulin isolated from immune rabbit serum. Counts were made of total cells and cells with nuclei exhibiting fluorescence. Under the conditions employed at present, only a small proportion (l-10%) of mouse embryo cells treated with polyoma virus, even at input multiplicities of 1000, are infected, if virus production is the criterion of infectivity. This difficulty has been encountered by other workers (Winocour and Sachs, 1960). However, it is clear from the data presented in Table 1 that essentially the same number of cells with fluorescing nuclei appeared in cultures incubated with and without FUdR, although no infectious virus was formed in the presence of the analoguc. It may be concluded, therefore, that polyoma antigen is synthesized under conditions in which no DNA and no infectious virus are made.
Electron
Microscopic
Studies
It was reported by Reissig and Kaplan (1962) that when chick embryo cells infected with pseudorabies virus were incubated with fluorouracil, characteristic viruslike particles were formed. The observation made in the foregoing experiment that in the presence of FUdR, nuclei exhibiting fluorescence appeared in infected cultures suggested that polyoma-like particles might be formed in the presence of this inhibitor. lMouse embryo cultures were infected with polyoma T and incubated in the prcscnce and absence of FUdR. Examination of cell preparations with the electron microscope (see Materials and Methods) after periods of 2448 hours, revealed that particles morphologically characteristic of polyoma virus (Howatson and Almeida, 1960) were present in the nuclei of cells of control and inhibited cultures (Fig. 4). The nuclear membrane and cytoplasmic vesicles are seen on the right edge of Figs, 4A and 4B. The characteristic polyoma-like morphology of particles formed in the presence of FUdR is seen in Fig. 4C. Such observations have been made with freshly-frozen material and with preparations embedded in methacrylate prior to sectioning. Quantitative Particles FUdR
Estimate of Polyoma-like Formed in the Presence of
In addition to the qualitative evidence obtained above for the formation of polyoma protein in the absence of DNA synthesis, a quantitative estimate of such protein formation was sought. One approach to this
TABLE
1
DETECTION BY FLUORESCENT ANTIBODY STAINING OF MOUSE EMBRYO CELLS CVNTAINING POLYOMA ANTIGEN IN INFECTED CULTURES INCUBATED IN THE PRESENCE AND ABSENCE OF FUdR Expt. no. 1
2
Period of incubation (hr)
Per cent fluorescent nuclei
PFU/ml - FUdR
+FUdR
- FUdK
+FUdR
0
3.0 x 10”
-
-
24 36
3.9 x 106 3.9 x 106
8.2 X lo4 5.8 x 104
5.4 8.3
5.7 10.9
0 48
2.5 X lo4 2.9 x 106
9.5 x 103
5.3
4.0
FIG. 4. Electron micrographs of the nuclear region of mouse embryo cells infected with polyoma T virus in the presence and absence of FUdR. Preparations shown in (A) and (B) wvc:re embedded in methacrylate and thin-sectioned. The preparation shown in (C) was frozen at -20°C and sectioned just prior to staining in phosphotungstic acid. A. Control culture, incubated wkhout FUdR. Magnification: X 50,000. B. Culture incubated with FUdR. Magnification: X 50,000. C. Similar to (B). Magnification: X 240,000.
problem was to count the numbers of viruslike particles formed in the presence and absence of FUdR. Mouse embryo cultures were infected and incubated with or without analogue for 32 hours. The culture lysates were spun at 8000 9 for 30 minutes to sediment cell debris. The supernatants were then centrifuged at 40,000 9 for 1 hour. The resulting pellets wcrc resuspended in PBS, and the suspensions were spun again at 8000 and 40,000 g as above. The pellets were taken up in 2 ml PBS and subjected to sonic vibrations at 10,000 kilocycles per second for 2 minutes. A volume of the suspensions was mixed with a known concentration of latex part,icles. These mixtures were treated with phosphotungstic acid and examined with the electron microscope. Count’s were made of latex particles and pnrticlcs with characteristic polyoma morphology and size. From these count.s was calculated the number of such part,icles formed in cultures incubated in the presence and absence of FUdR (Table 2). Although the plaque-forming units prescnt in the control culture after 32 hours
TABLE
2
QUAP*TIT,\TIVE I~ETERMINATION OF POLYOM.\-LIKE IN INPS~TICLES FORTNED AFTER 32 HOURS FECTED MORSE EJIBRYO CELLS INCUB.YTED IN THE PRESENCE IND ABSENCE OF FUdR.
Particle counW
Test material
PFU/ml
Control
4.90 x 108 1.36 x 109 1.03 X 103
9.5 x 106
8.10 4.90 1.76 1.52 1.31 1.58
1.8
Plus FUdR
Zero-time
culture
x x x X X x
No part.icles tectable
10” 108 IOY 10y
x
103
109
109 de-
2.1 X 104
a Data presented here were obtained using different sanlples prepared from one experiment.
cxcceded those present in the inhibited cultllre by a factor of almost 101, the number of virus-like particles in both cultures was t,he sa111e.
374
SHEININ
Measurement of Polyoma Antigen Formed in the Presence of FUdR by SerumBlocking Power A second and more specific quantitative determination of the amount of polyoma protein formed in the presence of FUdR was made using serum-blocking power (SBP) as criterion. The use of SBP for the study of noninfectious viral protein was introduced by De Mars (1955). He demonstrated that protein of bacteriophage T2, present as noninfectious virus, can be accurately measured by its ability to compete with intact phage for phage-specific antibody. The SBP of infected cultures incubated for 36 hours in the presence of FUdR, was compared with that of control cultures in which infectious virus was formed. Portions of the control cultures, harvested after 36 hours and frozen and thawed 3 times, were subjected to ultraviolet light (2537 A, 5 ergs/ mm”/sec) for 2 hours at approximately 10”. Such treatment, results in the loss of the plaque-forming ability of polyoma virus (G. F. Whitmore, 1963, personal communication). In the experiments described herein infectivity of the virus lysates was reduced by at least a factor of 106. However, the TABLE
3
MEASUREMENT OF SBP IN POLYOMA-INFECTED MOUSE EMBRYO CULTURES INCUBATED IN THE PRESENCE AND ABSENCE OF FUdR
Relative concentration of preparation
1 50 loo 200 500 1000 0
SBP! measured as the fractional survival of test virus, incubated with a standard immune serum, of the following: c-UV”
Fb
0.009
0.006 0.006 0.04 0.02 0.02 1.0 0.008
0.01 0.02 0.04 1.0 0.008
a C-UV, Control culture was incubated in the absence of FUdR. It produced infectious virus. For SBP assay, it was inactivated with ultraviolet light. b F, Culture incubated in the presence of FUdR.
haemagglutinating capacity and the particle morphology of the virus preparations remained unaffected. Preparations treated with ultraviolet light exhibited SBP. A comparison of the SBP power of control cultures containing infectious virus which were first inactivated by ultraviolet light, with t,hat of cultures prepared in the presence of FUdR, is shown in Table 3. The data show that approximately the same amount of serum-blocking material was formed in the presence and absence of FUdR, suggesting that the absence of DNA synthesis in the inhibited cultures has not interfered with the formation of specific polyoma protein. DISCUSSION
The presence of FUdR in cultures of mouse embryo cells infected with polyoma virus for up to 17l/2 hours has no effect on the subsequent time course of virus formation when the inhibitor is removed. It is therefore likely that viral DNA is synthesized very late during the latent period, i.e., no more than 30 minutes prior to the formation of intact virions. This conclusion is supported by the finding of R. Weil (1962, personal communication) that infectious DNA cannot be isolated from polyomainfected cells at any time prior to that at which intact virus first begins to appear. It is also indicated by the observation (Williams and Sheinin, 1961), that viral DNA, detected by acridine orange staining and deoxyribonuclease resistance, cannot be observed in the nuclei of infected cells before 18-20 hours after infection. The fact that FUdR-inhibition can be reversed up to 17l% hours with no effect on subsequent virus formation suggests that all the processes involved in polyoma reproduction, except replication of viral DNA, can proceed in the absence of synthesis of DNA, and in particular of viral DNA. Whereas FUdR prevents the synthesis of polyoma DNA,2 it does not appear to interfere with the formation of specific viral protein. All the available evidence indicates ’ Preliminary experiments indicate that particles formed in polyoma-infected cells in the presence of FUdR contain little or no nucleic acid.
FUdR
AND
POLYOMA
that the amount of viral protein formed in inhibited cultures is the same as that made in uninhibit,ed cells. Thus it seems likely that the original genetic material which enters the cells is able to direct the formation of all the new virus protein molecules formed. The formation of specific viral protein in the presence of FUdR has been reported for a number of DNA-containing viruses (Volkin and Ruffilli, 1962; Tanami et al., 1961; Salzman et al., 1963; Shatkin, 1963). These findings indicate that the replication of virus DNA may not necessarily be correlated with the other functions of this DNA in infected cells. There is, to date, one exception to the above observations. It was reported by Flanagan and Ginsberg (1962) and by Kjellen (1962) that adenovirus protein is not formed in the presence of FUdR. The significance of this finding is not clear, but may be related to the formation of a nonviral saline-soluble DNA associated with adenovirus infection (Ginsberg and Dixon, 1961). The demonstration that in the absence of DNA formation both polyoma antigen and morphologically characteristic viruslike part,icles are produced, indicates that the polyoma protein formed must be essentially the same at the level of primary and tertiary structure as that in infectious virus. Casper and Klug (1962) suggested that viral morphology was largely a function of the protein and that once formed, this protein could take up its characteristic morphological structure without linkage to DNA. This prediction has been borne out by the demonstration that virus-like particles are formed in the absence of DNA formation as a result of infection by polyoma virus and of pseudorabies virus (Reissig and Kaplan, 1962). ACKNOWLEDGMENTS The author’s grateful thanks go to Mr. Jim Morrison. His careful and thoughtful technical assistance is an essential part of the work reported. The critical comments of Dr. Louis Siminovitch throughout the course of this work have been most welcome. Mrs. June D. Almeida cheerfully coped with the electron microscopic studies.
375
FORMATION
Mr. Robert Escoffery prepared the fluoresceincoupled polyoma antiserum and carried out the fluorescent antibody labeling. This work was aided financially by grants from the National Cancer Institute of Canada, and by the U.S. Department of Public Health, Grants #AI-04229(VR), and #CA-04964. REFERENCES ALMEIDA, J. D., and HOWATSON, A. F. (19631. A negative staining method for cell-associated virus. J. Cell Biol. 16, 61&620. CASPAR, D. L. D., and KLUG, A. (1962). Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27, l-24. COHEN, S. S., FLAKS, J. G., BARNER, H. D., LOEB, M. R., and LICHTENSTEIN, J. (1958). The mode of action of 5-fluorouracil and its derivatives. Proc. Natl. Acad. Sci. U. S. 44,1004-1012. COONS, A. H. (1956). Histochemistry with labelled antibody. Intern. Rev. Cytol. 5, 1-23. DE MARS, R. I. (1955). The production of phagerelated materials when bacteriophage development is interrupted by proflavine. Virology 1, 83-99. DIMAYORCA, G. A., EDDY, B. E., STEWART, S. E., HUSTER, W. S., FRIEND, C., and BENDICH, A. (1959). Isolation of infectious deoxyribonucleic acid from SE polyoma-infected tissue cultures. Proc. Natl. Acad. Sci. U. S. 45, 1805-1808. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Eqcptl. h4ed. 99, 167-182. FLANAGAN, J. F., and GINSBERG, H. S. (1962). Synthesis of virus-specific polymers in adenovirusinfected cells: effect of 5-fluorodeoxyuridine. J. Exptl. Med. 116, 141-157. GINSBERG, H. S., and DIXON, M. K. (1961). Nucleic acid synthesis in Types 4 and 5 Adenovirus-infected HeLa cells. J. Exptl. Med. 113, 283-299. HEIDELBERGER, C. ( 1961) Nucleic acid synthesis and mechanism of action of fluoropyrimidines. In “Biological Approaches to Cancer Chemotherapy” (R. J. C. Harris, ed.), pp. 47-58. Academic Press, New York. HOWATSON, A. F., and ALMEIDA, J. D. (1960). Observations on the fine structure of polyoma virus. J. Biophys. Biochem. Cytol. 8, 828-834. KAHAN, F. M. (1960). Purification and measurement of microgram amounts of radioactive nucleic acids and proteins from animal cells in tissue culture. Anal. Biochem. 1, 107-126. KJELL~N, L. (1962). Effect of 5-halogenated pyrimidines on cell proliferation and adenovirus multiplication. Virology 18, 6670.
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PAHKEH, R. C., CASTOR, I,. N., and MCCULLOCH, E. A. (1957). Altered cell strains in continuous culture: A general survey. Spec. Publ. N. Y. Acad. Sci. 5, 303-313. REISSIG, M., and KAPLAN, A. S. (1962). The morphology of noninfective pseudorabies virus produced by cells treated with 5-fluorouracil. T/idogy 16, 1-8. SALZMAK,N. P., SHATKIN, A. J., and SEBHING, E. D. (1963). Viral protein and DNA synthesis in vaccinia virus-infected HeLa cell cultures. l’irology 19, 542-550. SHATKIN, A. J. (1963). The formation of vaccinia virus protein in the presence of 5-fluorodeoxyuridine. Virology 20,292-301. SMITH, J. D., FREEMAN, G., VOGT, M., and DULBECCO, R. (1960). The nucleic acid of polyoma virus. Virology 12, 185-196. SHEINIR., R. (1961). A rapid plaque assay for polyoma virus. Virology 15, 85-87.
SHEINIK, R. (1962). Procedures for the purification of polyoma T virus. Virology 17, 426440. SPIEGELMAN, S. (1957). Nucleic acids and the synthesis of proteins. Symp. Chem. Basis Heredity, Baltimore, 1958. Johns Hopkins Univ. McCollumPratt Inst. Contrib. 153, 232-267. TAKAMI, Y., POLLARD, M., and STARR, T. J. (1961). Replication pattern of psittacosis virus in a tissue culture system. Virology 15, 22-29. VOLKIN, E., and RUFFILLI, A. (1962). Dissociation of macromolecular synthetic processes in T2infected Escherichia coli. Proc. Natl. Acad. Sci. U. S. 48, 2193-2200. WILLIAMS, M. G., and SHEIKIN, R. (1961). Cytological skdies of mouse embryo cells infected with polyoma virus, using acridine orange and fluorescent antibody. Virology 13, 368370. WINOCOUR, E., and SACHS, L. (1960). Cell-virus interactions with polyoma virus. I. Studies on the lytic interaction in the mouse embryo system. Virology 11, 699-721.