Effect of polyoma virus on the replicative mechanism of mouse embryo cells

WROLOGY9.3, 73-84 (1965)

Effect of Polyoma Virus on the Replicative Mechanism of Mouse Embryo Cells ~ ROSE S H E I N I N AND P A T R I C I A A. Q U I N N The Ontario Cancer Institute, 500 Sherbourne Street, Toronto, and The Department of Microbiology, University of Toronto, Ontario, Canada Accepted January 20, 1965

TSPI polyoma virus elicits virus replication in essentially 100% of mouse embryo cells infected at high input multiplicity. Using this system it has been demonstrated that polyoma virus prevents mitosis in mouse embryo cells and that it inhibits ~he synthesis of host cell DNA. This inhibition appears not to be due to extensive destruction of host DNA template. INTRODUCTION

before virus formation (Sheinin, unpublished), which occurs in the nucleus (Henle et al., 1959). However, little is known about the biochemical events which occur early during the eclipse period of virus replication; nor is much known about the effects of polyoma multiplication on host cell metabolism. This lacuna in knowledge can be attributed mainly to one serious experimental problem. Only a small proportion of cells treated with polyoma replicate the virus, even when high input multiplicities of infection are used (Winoeour and Sachs, 1960; Weisberg, 1963; Sheinin, 1964). Recently we have found that almost all mouse embryo cells produce virus when infected, at high nmltiplicity, with a particular small-plaque variant of polyoma T, T S P I (Stanners, 1963). This observation has permitted the investigation of the effect of polyoma virus on host cell mitotic activity and on host D N A synthesis. The results of these studies are reported here.

A number of DNA2-eontaining animal viruses are known, the formation of which occurs in the nucleus of infected cells. This phenomenon of nuclear maturation is of obvious interest with respect to the interrelationship between cell and virus multiplieation, and between the synthesis of ceil D N A and that of the virus DNA. Of those D N A viruses which are formed intranuelearly, polyoma virus is of special interest because although this virus is cytoeidal in certain conditions, it can also cause genetic transformation in cells which it infects (Vogt and Dulbeeeo, 1960; Medina and Sachs, 1961). Of the events which occur during polyoma replication, it has been shown that the D N A is synthesized from constituents of the soluble pool (Sheinin, unpublished) at the end of the latent period just prior to virion maturation (Sheinin, 1964). Capsid protein synthesis appears also to take place just This work was supported by grants from the L*.S. Department of Public Health (no. AI-04229 (VR)) and (no. CA-04964), and from the National Cancer Institute of Canada. Abbreviations: DNA, deoxyribonucleic acid; PBS, phosphate-buffered saline (Dulbeeco and Vogt, 1954); PFU, plaque forming units; TdR, thymidine; TdR-H ~, tritiated thymidine; dpm, disintegrations per minute.

MATERIALS AND METHODS Cultivation of Mouse Embryo Cells

Procedures for the cultivation of mouse embryo cells have been described (Sheinin, 1961, 1962). 73

74

Preparation of Polyoma Virus

SHEININ

AND QUINN

on degreased coverslips (22 mm square), in Ordinary stocks of the small-plaque plastic petri dishes (10 X 35 mm) were used. variant of polyoma T, TSPI (Stanners, These cultures were prepared by putting 1963) were prepared by procedures described down approximately 8 X 105 primary cells earlier (Sheinin, 1962). However, to obtain per dish. The cultures were used 18-24 hours preparations containing a high concentration later, when the cells were almost confluent. of TSPI virus, infected cultures were The medium was removed, and 0.1 ml of virus suspension was added to the centre of harvested in the following way. Secondary cultures of mouse embryo cells each eoverslip. This volume spread to cover were infected with virus from stock lysates almost the entire coverslip. Unless otherwise of TSPI, at a multiplicity of 10-100 PFU noted, cells were infected with a multiplicity per cell. The cultures were incubated at 37 ° of about 1000-2000 PFU/cell (calculated on for 6-7 days, at which time the eytopatho- the basis of the number of cells on the covergenie effect of the virus on the cells was first slip). The cultures were then incubated for detectable. The cell sheets were shaken from 90 minutes at 37 ° in an incubator flushed the glass and the cells were harvested by with 5 % CO2:95 % air. Medium (2 ml) was eentrifugation at 2 ° at 800 g for 20 minutes. added, and the cultures were reineubated. The cells were washed and resuspended in For all other studies the cells were grown in PBS at a concentration of approximately 2-ounce Brockway bottles as already de10~ cells per millilitre. The cells were frozen scribed (Sheinin, 1961). and thawed three times and were subjected Swelling and Fixing of Cells for Mitotic Index to ultrasound at 10,000 ke per second for 10 Measurements, for A utoradiography and minutes. This suspension of cell debris was for Immunofluorescence Studies incubated at 37 ° for 24 hours with neuraPreparation of cells for studies of mitosis, minidase (1 unit/ml 3) to destroy inhibitory and DNA synthesis, by autoradiography, mucoproteins (Crawford, 1962). The di- required great care in order that the cells not gested suspenson was centrifuged at 800 g be lost from the surface of the eoverslips. for 20 minutes, yielding the supernatant The cells were swollen essentially by the containing the virus. Immediately prior to procedure of Rothfels and Siminoviteh use, these supernatants were treated with (1958). Two millilitres of distilled water was ultrasound (as described above) to dissociate added dropwise to the medium in the petri aggregates of the virus formed during dishes. The cultures were left at room temstorage at - 20°C. perature for at least 10 minutes. The diluted medium was decanted and replaced with 2 Assay of TSPI Virus ml water. The cultures were again left for at TSPI polyoma virus was assayed by a least 10 minutes. The water was decanted, modification of the plaque assay already and the eoverslips were removed from the described (Sheinin, 1961). At 6 days after dishes and drained. The cells were allowed infection, 2 ml of agar-medium overlay was to air dry completely. added to the initial overlay. Plaque counts For fixation of the cells, the coverslips were usually made on the ninth or tenth day were placed in glass petri dishes. The after infection. fixative was added dropwise to each coverInfection of Mouse Embryo Cells with TSPI slip and left on the cells for 10 minutes. For mitotic index measurements the cells were Polyoma Virus fixed in Carnoy's solution and were then For the autoradiographic studies and those stained with oreein solution (2% w / v in on mitotic index, secondary cultures of acetic acid, 50% v/v). For immunoftuomouse embryo cells, growing exponentially rescenee studies the cells were fixed with acetone. Polyoma-infeeted cells fixed in s One u n i t of n e u r a m i n i d a s e is t h a t a m o u n t of acetic acid solutions did not react with e n z y m e w h i c h releases 1 ~g of N - a c e t y l n e u r a m i n i c antiserum to the virus. Cells fixed in either acid f r o m a s t a n d a r d g ] y c o p r o t e i n s u b s t r a t e in 15 way could be used for autoradiography. m i n u t e s at 37 ° .

POLYOMA AND HOST DNA SYNTHESIS

Measurement of Mitotic Index Cells in all stages of mitosis were counted for the determination of mitotic index. At. least 1000 cells were counted in duplicate coverstip preparations. The mitotic index is expressed as the number of cells in mitosis per 100 cells counted.

Measurement of Infectious Immunofluorescence

Centres by

The procedure for staining polyoma virus in infected cells (fixed with acetone), with l~uorescein-conjugated antipolyoma ~,-globulin has been described (Sheinin, 1964). The infectious centres originating as a result of polyom~ infection were scored in terms of cells with fluorescing nuclei. (In other experiments we have established that the nmnber of infectious centres measured in this way is the s~me as that determined by plaque assay if cells are plated on mouse embryo cells immediately after infection.) Total cell counts and counts of cells with fluorescing nuclei were made on five randomty-selected areas on duplicate coverslips. Between 500 and 1000 cells were counted per eoverslip.

Assessment of DNA Synthesis By autoradiography. To examine DNA synthesis in control and polyoma-infected coverslip cultures by autoradiography, the cells were exposed to a pulse of tritiated thymidine, in the following way. The growth medium was removed and replaced with 1.5 ml of the following medium: CRML 1066 (Parker, 1961) minus thymidine and minus coenzymes, supplemented with TdR-I-t 3 (1.3 raM; 10 ~e/ml). The cells were incubated with the radioactive medium for 1 hour at 37 ° . They were then swollen, fixed, and stained. The coverslips were then dipped in nuclear track emulsion and processed by standard autoradiographic procedures, after exposure for 5-7 days. Counts were made of cells containing labelled nuclei: at least 1000 ceils were counted in duplicate coverslip preparations.

By isolation of radioactively gabeled DNA. Secondary cultures of mouse embryo cells were grown in 2-ounce Brockway bottles in the following medium: CRML 1066 minus

75

TdR supplemented with dialyzed foetal calf serum (5% v/v) and TdR-H a (0.03 ~M, 0.2 /~c/ml). When the cells had become contiguous, the medium was decanted and the ceils were washed three times with 1066. Control cultures were handled as were the infected ones, except that they received no virus. Test cultures were infected with TSPI virus as described above. All cultures were then incubated in complete nonradioactive medium. For harvest of the DNA, the growth medium was decanted and the cells were washed three times with PBS. The cells were removed from the glass by trypsinization (Sheinin, 1961), and an aliquot was removed for determination of cell numbers. The remaining cell suspension was chilled in an ice bath. Five millilitres of cold perchloric acid (5 % v/v) was added to precipitate the maeromolecules. It had been established that under the experimental conditions employed the TdR-H a label appeared only in DNA. The precipitate was collected on Millipore filters and was washed twice with 5 ml cold perchloric acid. The filter was sucked dry and was then transferred to a vial for measurement of radioactivity in a scintillation counter using nonaqueous fluid (Arnold, 1961). Each result reported is the average obtained from three different cultures.

Chemicals and Reagents Unless otherwise noted, all reagents used were BDH, AnalaR grade. Orcein was purchased from Matheson, Coleman, and Bell; tritiated thymidine from New England Chemicals Corporation; and the 4,5diphenyloxazole and 1,4-bis-2-(4-methyl-5phenyloxazolyl)benzene, used for scintillation counting, from Packard Instruments Company. Ilford Nuclear Research Emulsion used was in the gel form, G5. The Millipore filters were obtained from the Millipore Corporation. Neuraminidase was an extract of Vibrio eholerae purchased from General Biochemicals. RESULTS

Properties of the Experimenta~ System One of the maior difficulties which has beset studies on the interaction of polyoma

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SHEININ AND QUINN

virus and host cells has been the inability to obtain more than a very small proportion of such cells as infectious centres, even when very high input multiplicities of infection were used. This problem has been overcome using the T S P I variant of polyoma T. When mouse embryo cells are infected at a multiplicity of at least 103 PFU/cell, most, if not all, of the cells were found to produce virus (Fig. 1), as indicated by the observation that at 16-18 hours the nuclei of 100 per cent of the cells fluoresced, when treated with fluorescein-conjugated anti-

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serum to polyoma virus. Both virus formation, as measured by infectious virus, or viral protein formation, as measured by immunofluorescence, were first detected at about 14 hours. Most of the cells were producing virus protein by 16 hours, but virion production proceeded until approximately 36 hours.

Winocour and Sachs (1960) found that with four different plaque isolates of SE polyoma virus (Stewart et al., 1958) increasing numbers of virus-producing mouse embryo cells could be obtained by increasing the input multiplicity of infection; at concentrations of about 2 X 103 PFU/cell a majority of cells scored as infectious centres. We have experienced considerable difficulty in routinely obtaining more than 10% of cells as virus-producers, even when using as IO0 many as 104 PFU/cell, when working with the wild-type polyoma T, or a large-plaque mutant derived from it. For study of the effect of input multi,o7 o~ plicity of TSPI virus on infectious centre ._c formation, coverslip cultures of mouse # embryo cells were infected (as described in Materials and Methods) with varying ,o6 amounts of virus and were incubated for 20 hours. The cultures were processed and > examined for virus-producing cells by the technique of immunofluorescence. The data id o shown in Fig. 2 indicated that the number of cells producing polyoma antigen is related to the amount of virus added over the range of 30-103 PFU/celI. Other experiments ~o4 revealed that 100% of the cells can be

FIG. 1. Multiplication of TSPI polyoma virus in mouse embryo cells. Mouse embryo cells in secondary culture were grown in 2-ounce Brockway bottles or on coverslips, for measurement of virion production and infected cells, respectively. The cells were infected and harvested at the time intervals noted, as described under Materials and Methods. For virion assay the cultures were frozen and thawed three times, were incubated at least 24 hours at pH 8-8.4, were subjected to ultrasound (10,0fl0kilocycles per second) for 5 minutes, and were assayed by plaque assay. Infected cells were measured by immunofluorescenee in the nucleus, t • Polyoma virus; O-----O infected cells.

infected routinely using input nmltiplicities of 103 to 5 X 103 PFU/cell.

Effect of Polyoma Virus on Mitosis in Mouse Embryo Cells From the experiments described above it is clear that all mouse embryo cells can be infected with the T S P I variant of polyoma virus. It is therefore possible to study the effects cf such infection on the host cell. Since polyoma D N A (Williams and Sheinin, 1961) and protein (Henle etal., 1959) appear to be formed in the nucleus, it was of interest to examine the effect of the virus on normal nuclear processes, for example, mitosis. An

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Multiplicity of infection(PFU/cell) Fio. 2. t~elationship between t h e m u l t i p l i c i t y of infection and the n u m b e r of mouse embryo ceils producing polyoma protein. Mouse embryo cells on eoverslips were infected, as already described, w i t h v a r y i n g c o n c e n t r a t i o n s of T S P I virus. T h e cells were h a r v e s t e d after 18 hours of i n c u b a t i o n a n d were processed and examined b y the t e c h n i q u e of immunofluoreseence, for the numbers of cells producing virus.

effect on mitosis was to be expected, since we had found that infection with a largeplaque illUtrant led to death of a majority of mouse embryo cells, as measured by the inability of infected cells to form a macroseopie colony (Sheinin, unpublished). Coverslip cultures of mouse embryo cells were infected with TSPI, at an input

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FIG. 3. Effect of T S P I virus on the mitotic index of mouse embryo ceils. Mouse embryo cells on eoverslips were infected and processed as described u n d e r Materials and Methods. At the time intervals noted, t h e n u m b e r of infected cells a n d of the cells in mitosis were determined. @ - - - - @ Infected cells as d e t e r m i n e d b y immunofluorescence in the nucleus; X - - - - X mitotic index of noninfected cells; O © mitotic index of infected cells.

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Fie. 4. Effect of polyoma virus on D N A synthesis in mouse embryo cells, as measured b y the incorporation of t r i t i a t e d t h y m i d i n e into D N A by the technique of a u t o r a d i o g r a p h y . Cultures of mouse embryo eells on coversIips were infected, incubated, and processed at the n o t e d time intervals, as described u n d e r Materials a n d Methods. a. Control culture, b. Polyoma-infeeted culture.

78

SHEININ AND QUINN

multiplicity of about 2 X 103 PFU/cell. After the intervals indicated in Fig. 3, duplicate eoverslips were processed for (a) measurement of infectious eentres by immunofluorescence and (b) measurement of mitotic index. The data obtained show that by 4 hours after infection the mitotic index of infected cells had been considerably reduced below that of uninfected cells. B y 10-12 hours few, if any, mitotic figures were observed in infected cultures. T h a t this inhibition of mitosis is a function of the virus is indicated by the finding that an extract of uninfected cells had no effect on cell division in mouse embryo cells. Nor was there any effect on mitosis when cells were treated with virus inactivated with ultraviolet light, to the extent of a drop in titre of 105 P F U / m l .

It may be of significance that the time at which the mitotic activity of infected cells reaches its minimum coincides with the time at which virus antigen formation is first detectable.

Effect of Polyoma Virus on DNA Synthesis in Mouse Embryo Cells Since changes in the nucleic acid and nucleoproteins had been reported in cells infected with polyoma virus (Allison and Armstrong, 1960; Love and Rabson, 1961; Williams and Sheinin, 1961), it was of special interest to examine the effects of virus infection on DNA replication of the host cell, which also occurs in the nucleus. DNA synthesis was examined, by the technique of autoradiography, in control and polyoma-infected mouse embryo cells. This

FIG. 5. Autoradiographs of normal and polyoma-infeeted mouse embryo cells exposed to a pulse of tritiated thymidine, a. Normal culture, b. Polyom~-infeeted culture which received TdIK-H~ 19-20 hours after infection of (b) cultures. Magnification: X969.

POLYOMA AND HOST D N A SYNTHESIS

79

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technique, in combination with immunofluorescent staining, has been employed by Minowoda (1964) to demonstrate what is probably polyoma D N A synthesis in Xirradiated cells. It was first established (Fig. 4a) that in cultures of mouse embryo cells growing logarithmically on coverslips, 25-30 % of the cells were synthesizing DNA; i.e., 25-30% of the cells subjected to a pulse of tritiated thymidine were found to have labelled nuclei. This proportion fell off gradually as cell multiplication declined in the course of the experiment. Since an asynchronous population of cells was used in these studies the labelled ceils would be expected to present two major kinds of appearance. Those cells which were present in the phase of D N A synthesis for only a short period during the pulse, would contain little T d R - H a and would appear as

"lightly labeIled." Those cells synthesizing DNA for a considerable part of the pulse period would contain more T d R - H 3, and would appear as "heavily labelled." For the purposes of comparing normal cultures with those infected with polyoma virus, the "heavily labelled" cells are defined as those the nuclei of which appear as a solid black mass; the "lightly labelled" as those, the nuclei of which are overlaha b y separated grains (Fig. 5a). Using these criteria it was foulld that of the labelled cells in uninfected cultures, 80 90% were "heavily labelled" (see Fig. 4a). Mouse embryo cells infected with polyoma virus presented a pattern of D N A synthesis similar to that of uninfected cells for approximately 12 hours (Fig. 4b). During this period about 25 % of the cells became labelled when subjected to a pulse of T d R - H s. Of these labelled ceils, 88% appeared as

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"heavily labelled." A striking change in the labelling pattern was observed at 12 hours. The fraction of cells synthesizing DNA increased rapidly to reach a maximum at 20 hours. Most of these cells were not "heavily labelled." In fact, of the 88 % of labelled cells present at this time, only 3% were so labelled (Fig. 4b). It is clear that during the interval between 12 and 20 hours the pattern of labelling in infected cultures changed to one in which more and more cells were synthesizing DNA in amounts producing a "lightly labelled" appearance (Fig. 5). The change in the pattern of DNA synthesis in polyoma-infected cells occurred at the time at which virus-producing cells (as detected by immunofluorescence) began to appear in the culture. The numbers of cells synthesizing DNA in the light pattern and the number of cells producing viral antigen increased after 12 hours to reach 88 and 87 %, respectively, of the cell population at 20 hours. The close correspondence between the kinetics of appearance, and the numbers, of "lightly labelled" cells and those producing viral antigen, suggests that the DNA synthesis observed in these cells is polyoma DNA synthesis. Preliminary experiments in which the formation of viral antigen and DNA synthesis have been examined in the same cells have provided confirmation for this conclusion. The change in DNA labelling pattern was not observed if cells were treated with control extracts, or with TSPI suspension inactivated with ultraviolet light. Thus this effect on the DNA synthesis of the host cell is indeed a function of the polyoma virus.

amining the fate, during infection, of cell DNA heavily prelabelled with TdR-H 3 prior to treatment with TSPI virus. If degradation of host DNA is extensive, and proceeds to soluble products which are not utilized for viral DNA synthesis, then it would be expected that the proportion of cells labelled should remain constant after infection, but the degree of labelling should decrease continually until such time as label l O O II~

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Effect of Polyoma Virus on the D N A of Mouse Embryo Cells One of the remarkable features of the above observations is that concomitant with the onset of viral DNA and protein formation, the synthesis of host DNA comes to a halt. One may ask by what mechanism the replication of cell DNA synthesis is prevented. An obvious hypothesis is that the cell DNA template is degraded as a result of polyoma infection. This hypothesis can be tested by ex-

FIG. 6. Effect of polyoma virus on preformed mouse embryo cell D N A as measured by autoradiography. Cultures were grown on coverslips in medium containing T d R - H 3. The cells were infected with T S P I virus and processed for autoradiography and immunofluorescence as described under Materials and Methods. Counts were made of the number of infected cells with fluorescing nuclei, (X X ) ; of the labelled nuclei ill control cultures (O O); and of those infected w i t h polyoma virus ( 0 O). a. N u m b e r of labelled cells, b. E x t e n t of labelling.

POLYOMA AND HOST D N A S Y N T H E S I S

is no longer detectable. If the soluble products are incorporated into polyoma DNA, without dilution, then one would again expect the proportion of labelled cells to remain constant during infection, however the degree of labelling should decrease prior to the time of viral DNA synthesis and should then remain constant or increase again as virus DNA is made. To examine these possibilities, mouse embryo cells (on eoverslips) were grown for 24 hours prior to infection in TdR-It acontaining medium, to obtain cells with radioactive DNA. The cells were then infected with TSPI and after the intervals indicated in Fig. 6, the number of labelled ceils in the cultures, and the degree of their labelling, was determined. The results of such a study showed (Fig. 6a) that 100 % of the cells were labelled at the start of the experiment and remained so throughout the period of study. What is more important is that 100% of these cells were "heavily o 60,000

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FIG. 7. Effect of polyoma virus on the D N A of mouse embryo cells. Secondary cultures of mouse embryo cells were grown in TdR-H~-eontaining medium and were infected where indicated, as described under Materials and Methods. At the intervaIs noted the eelIs were harvested. Counts were made of the cell numbers present. The TdR-H3-1abelled D N A was precipitated with cold perehlorie acid. The labelled D N A was collected on a Millipore filter, and its radioactivity was determined. The results of triplicate analyses are presented as the TdR-H~ in the D N A of control cultures, I - - - - O ; and those infected w i t h polyoma virus, O - - ©.

81

labelled" at time zero (Fig. 6b), but remained so labelled even at 22 hours when all the cells were producing polyoma virus, as measured by antigen formation. These observations suggested that no extensive breakdown of preformed cell DNA had occurred as a result, of TSPI infection. To investigate this more directly and more accurately, the above experiment was repeated; however, at the intervals noted in Fig. 7, the DNA was extracted and its radioactivity was measured. The evidence obtained indicated that little or no mouse embryo cell DNA was lost during the period of polyoma infection. It should be pointed out that the polyoma DNA formed per cell constitutes approximately 4 % of the total DNA of the cell. Thus if all the mouse cell DNA were degraded and only 4 % of this material was reineorporated into viral DNA, such a transfer should be readily detectable. DISCUSSION

Polyoma virus, which was known to multiply in the nucleus and to elicit a change in the nucleic adds of host cells, has now been shown to suppress mitosis in mouse embryo cells. This inhibition of cell division is an early function of the virus, being detectable by 4 hours after infection. This rapidly expressed effect of polyoma virus on mitosis is emphasized by comparing its action with that of other eytopathie viruses. Poliovirus has no effect on mitosis (or cell DNA synthesis) until virion produetion is well under way (Defendi, 1962). Reovirus actually stimulates mitosis, albeit aberrant (Dales, 1963), and may require initiation of the mitotic process for its development (Dales, 1963; Spendlove et al., 1963). Rabies virus can also multiply in certain cells without interfering with the mitotic process (Fernandes et al., 1963). These viruses, of which poliovirus and reovirus have been shown to contain RNA, are known to multiply in the cytoplasm and might therefore be expected to have no effect on the mechanisms operating in the nucleus. In the ease of DNA viruses, the effects are markedly different. Their action with respect to mitosis permits one to group them in two categories, (a) those which prevent

82

SHEININ AND QUINN

the cell from entering mitosis, and (b) those which interfere directly with the mitotic process producing mitotic arrest or chromosomal aberrations. Prevention of mitosis has been shown to occur with adenovirus (Prunieras and Chevalier, 1964), ectromelia virus (Kato et al., 1960) pseudorabies virus (Reissig and Kaplan, 1960), vaccinia virus (Kit and Dubbs, 1962), and polyoma virus. Interference with the actual process of mitosis and the production of altered chromosomes has been demonstrated with herpes virus (Roizman, 1961; Hampar and Ellison, 1963; Stich et al., 1964), measles virus (Nichols et al., 1962), and SV40 virus (Koprowski et al., 1962; IVIoorhead and Saksela, 1962; Shein and Enders, 1962). The autoradiographic studies reported here indicate that the synthesis of the DNA of polyoma TSPI virus commences after 12 hours and essentially parallels the formation of viral antigen. This observation is in accord with studies using 5-fluoro-2'-deoxyuridine (el. Sheinin, 1964), which indicated that TSPI DNA is synthesized just before virion maturation, which begins just after 12 hours (Sheinin, unpublished). Although approximately 30% of mouse embryo cells are synthesizing DNA at any time, in a logarithmically growing culture, essentially 100 % of these cells make polyoma DNA when infected (Fig. 4). It is clear, therefore, that in the 70% of cells not normally synthesizing DNA, polyoma infection must result either in the induction of new, or in the derepression of already existing, enzymes involved in the conversion of thymidine to DNA. It is to be hoped that future experiments will reveal which enzymes are specifically involved and whether they are host-controlled or virus-induced. A second function of polyoma virus has been uncovered in these studies, that being the inhibition of host cell DNA synthesis. This inhibition commences at approximately 12 hours after infection, apparently coincident with the time at which viral DNA synthesis occurs. This phenomenon is reminiscent of the situation observed with some bacterial viruses: infection of Escherichia coli B with the T-even bacteriophages results in a cessation of host DNA replication

(Kozloff, 1953). However, in the bacterial system the inhibition of host DNA synthesis occurs immediately after infection, whereas with polyoma virus, host DNA synthesis continues, apparently normally, until viral DNA synthesis commences. The difference in the time at which host DNA synthesis is inhibited in the two situations may be related to the fact that the bacterial DNA is degraded and utilized for virus DNA synthesis. Such host DNA breakdown does not appear to occur during polyoma infection. Polyoma DNA synthesis has been shown to proceed from pool constituents supplied by the medium (Sheinin, unpublished). The delay of 12 hours in the onset of synthesis of polyoma DNA and protein, and in the inhibition of host DNA synthesis, cannot be due to a delay in adsorption or eclipse of the virus. For we have shown that such eclipse occurs within one-half hour after infection. Furthermore, TSPI produces a suppression of mitosis, which is already detectable 4 hours after infection. It therefore seems likely that the expression of the polyoma genome requires a number of processes which occur during the first 12 hours of the eclipse period, but which in themselves do not interfere with host DNA synthesis. Whether the inhibition of host DNA synthesis is a necessary prerequisite for the commencement of polyoma DNA synthesis is not known. Nor is it known whether this inhibition is triggered by the initiation of virus DNA formation. However, the interrelationship between these two processes is of obvious interest with respect to the control of DNA synthesis. ACKNOWLEDGMENTS It is always a pleasure to thank Dr. L. Siminovitch for his constant critical contributions to the experimental work. The careful and cheerful assistance of Mr. Jim Morrison is gratefully acknowledged. The antisera to polyoma virus were prepared by Mr. Walter MacKinlay and their fluorescein-eonjugated derivatives were prepared by Mr. Robert Escoffery. REFERENCES A~LISON, A. C., and A~MST~O~G, J. A. (1960). Abnormal distribution of nucleic acid in tissue

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