The response of BHK21 cells to infection with type 12 adenovirus

VIROLOGY

The

39, 642-652

Response

I. Cell Killing

(1969)

of BHK21

Cells

and T Antigen

to Infection

Synthesis

as Correlated

WILLIAM Department

of Microbiology, New

with

Type Viral

12 Adenovirus

Genome

Functions’

A. STROHL

Rutgers Medical School, Rutgers, Brunswick, New Jersey 08903 Accepted

July

The State

University,

17, 1969

BHK21 cell killing by type 12 adenovirus (Ad12) has been shown (1) to be a viral genome function, (2) to require only one successfully infecting virus particle, and (3) to be induced with an efficiency somewhat greater than the initiation of viral early protein synt.hesis, measured as Ad12 tumor-specific antigen (T Ag). When the multiplicity of infection is increased beyond that required to initiate T Ag synthesis in 980/, of the cells, a persistent surviving fraction of cells is found, the magnitude of which is dependent on the extent and rate of cell multiplication during the first 24 hours after infection. Analysis of T Ag synthesis in clones produced by infected cells indicates that while infected cells can divide initially, the capacity to synthesize T Ag is not replicated, since the multiplying cells are all T Ag (-) even though they may be the offspring or sisters of T Ag (+) cells. While single T Ag (+) cells may persist for several days after infection, most of these detach from the coverslip within 2448 hours after infection and can no longer give rise to clones. The increased survival of cells infected at high multiplicity while in logarithmic growth cannot be accounted for only by dilution of the infecting genomes and is not due to a genetically resistant cell type in the population. INTRODUCTION

In the course of the work on transformation of BHK21 cells by type 12 adenovirus (Ad12), extensive killing of the infected cells was observed at high input multiplicities (Strohl et al., 1968; Strohl, 1969a). Similar observations have been noted in other reports of hamster cell transformation by adenoviruses (Casto, 1968). A second, related response to adenovirus infection of most species of cells has been the production of chromosomal breakage in those cells still capable of dividing some time after infection (Mackinnon et al., 1966). zur Hausen (1968b) demonstrated that the extent of chromo1 Supported by Public Health Service Research Grants AI-66011 and CA-68851. A preliminary report of some of these findings was presented at a meeting of the Federation of American Societies for Experimental Biology [Federation Proc. 27, 682 (1968)l. 642

somal fragmentation was related to the number of cells synthesizing the tumorspecific antigen (T Ag) and proposed that the chromosomal damage might be the cause of the cell death that followed Ad12 infection of hamster cells. Clearly, there exists an exceptional alternative to the death of infected cells-namely neoplastic transformation and the long-term survival of such transformed cells. In another report (Strohl, 1969a), it has been shown that among the rare survivors or their progeny, transformation occurred with surprising frequency (at least 40%). Therefore an understanding of factors influencing the outcome of infection-death or survivalwill inevitably point to those distinguishing features that facilitate transformation. The present paper explores in more detail the quantitative relations between Ad12 genome expression and cell killing and the

BHK

CELL

KILLING

modification of these relationships by the physiological state of the cell. The accompanying paper (Strohl, 196913) discusses the induction of cellular DNA synthesis by Ad12 infection of nonmultiplying cells, and its possible correlation with cell killing. MATERIALS

AND

METHODS

Cells. A culture of BHK21/13 cells (Stoker and Macpherson, 1964), 65 generations from original cloning, was obtained from Dr. Ian Macpherson, Glasgow, Scotland. A new clonal isolate, designated B,, (Strohl, 1969a), was selected, and a supply of the cloned cells was frozen at - 120”. For the experiments to be described, cells taken from this frozen stock were kept in logarithmic growth and were used between the 1st and 12th passage. Human embryonic kidney (HEK) cells were obtained commercially; they were used after 1 to 3 passages. Media. All cell culture was done in an enriched basal medium of Eagle, designated HT medium, as described in Rouse et al. (1966), except that fetal calf serum (FC) was used exclusively.. Cells to be cloned were suspended with a 0.025% solution of Viokase (Grand Island Biological Company, Grand Island, New York) in Ca2+- and Mg2+-free phosphate-buffered saline (PBS) containing 0.02 % ethylenediamine tetracetate (EDTA). Virus. Plaque-purified preparations of Ad12, strain Huie, were grown on HEK cells. The stock virus was prepared by concentrating the infected cells in a small volume of 0.01 M Tris buffer at pH 8.1 and disrupting the cells in a Raytheon 10 kc sonic oscillator. The suspension was clarified by low speed centrifugation and stored frozen at -70”. Such preparations contained from 1 to 5 X log plaque-forming units (PFU) per milliliter when assayed on HEK cell monolayers. Ad12 Plaque Assay. Samples to be assayed were diluted in PBS without serum, and allowed to adsorb to HEK cell monolayers for 2 hours at 37’; the monolayers were then overlaid with a nutrient agar medium similar to that described by Rouse et al. (1963), but enriched with 2X amino acid and vitamin concentrations, Eagle’s nonessential amino acid mixture (Eagle, 1959), and 10 % FC. tTV Irradiation of Adlb. Ad12 virus at

BY

Ad12

643

2 X log PFU/ml was clarified by centrifugation at 2500 rpm for 5 min, diluted 1:5 in PBS, and irradiated in open petri dishes with two 15-W germicidal bulbs at a distance of 6.5 cm. The virus suspension was agitated throughout the irradiation. ImmunojIuorescence technique. The cells to be tested for virus-specific tumor antigen (T Ag) by the indirect immunofluorescence technique were grown on coverslips and fixed for 10 min in prechilled (-70”) acetone at room temperature. After air-drying, the fixed cells were reacted with antiserum at 37”. T Ag was detected with a pool of hamster sera from animals hyperimmunized with cultured Adl2-induced hamster tumor cells (HT2) (Strohl et al., 1963, 1966). The fluorescein-conjugated caprine antihamster r-globulin was obtained commercially from Progressive Laboratories, Inc., Baltimore, Maryland. Appropriate controls with normal hamster serum and with uninfected BHK21 cells were included in all experiments. The staining procedures and the determination of frequency of cells synthesizing T Ag were done as described previously for Ad2 structural antigens (Strohl et al., 1966), except that the microscope fields were calibrated at each magnification, thus enabling the number of cells per culture to be calculated. Assay of cell survival by cloning in jluid medium. Cells to be cloned were counted in a hemacytometer, serially diluted in HT medium supplemented with 20% FC, and 0.2 ml of each dilution was seeded into each of a number of petri dishes containing the same medium. These were left undisturbed for 5-7 days, at which time uninfected cells had produced grossly visible colonies. All cultures were then fixed with methanol, stained with Giemsa stain, and grossly visible colonies counted. Clone survival was expressed as the ratio of the fraction of infected cells which formed colonies relative to the fraction of uninfected cells which formed colonies. The uninfected cell cloning efficiency usually fell between 15 % and 40% of the plated cells. In a few cases, the cells to be cloned were seeded into cultures containing 5 X 10’ BHK21 feeder cells which had been Xirradiated with 5000 r. The uninfected cell

644

STROHL

cloning efficiency on feeder cells was generally between 50% and 100 %. RESULTS

Correlation of Cell Killing Synthesis

with

T Antigen

The relationship between infecting virus dose and cell death was studied by infecting BHK21 cells with serial dilutions of Ad12 and assaying the infected cultures for survival of clone-forming capacity. Since adsorption of adenovirus to hamster cells is relatively inefficient (Rouse et al., 1966), the input multiplicity of virus was not considered to be a reliable measure of infection. The synthesis of early proteins (measured as Ad12 T Ag) seemed likely to be a more unequivocal indication of successful infection, and all measurements of cell killing were therefore related to the fraction of cells containing T Ag 24 or 48 hours after infection. Quantitaticm of the killing effect. Experiments designed to determine the basic relationships between multiplicity of infection (moi) and cell killing were carried out as follows. Petri dishes, with or without coverslips, containing approximately 1 X lo6 cells per plate were infected with serial dilutions of Ad12. After 2 hours’ adsorption at 37”, INPUT

cultures with coverslips were washed twice in HT medium without serum, fed with medium containing 0.2 % FC, and incubated at 37”. Cells on the coverslips were fixed at 24 and 48 hours after infection and examined for T Ag by the immunofluorescence procedure. The frequency of specifically stained cells was calculated with respect to the cell number in control cultures fixed at the time of infection. Uninfected cells multiplied no more than about 2-fold under these conditions of feeding (Strohl, 1969b). Cultures without coverslips were washed and suspended with Viokase immediately after the adsorption period, and then assayed for survival of clone-forming ability as described in Materials and Methods. Typical results are shown in Fig. 1. The frequency of cells synthesizing T Ag increased in direct proportion to the virus dose, indicating that the T Ag system could be used as a valid assay system for single, successful infections. It may be noted that the dose required to achieve an average of one T Ag inducing infection per cell (i.e., 63% of the cells with T Ag, by the Poisson expression, and hereafter referred to as 1 TAU) was about 14 PFU per cell. The same moi of Ad2 was found previously to be equivalent to 1

MOI (PFIJICELLI

FIG. 1. Relationship of T Ag synthesis to cell killing after infection of BHKPl cells with Ad12. Infection was carried out a.~ described in the text, and the frequency of cells synthesizing T Ag was determined by immunofluorescence staining. The curve drawn through the T Ag points is that of the theoretical l-hit relationship between virus dose (moi) and T Ag response. Clone survival represents the fraction of the control cell population retaining the ability to form grossly visible clones after Ad12 infection at the indicated moi. Open symbols: T Ag synthesis; closed symbols : clone survival. The circles and triangles refer to results from two different experiments.

BHK

CELL

0

x

O-21

r

I

2

KILLING

3 4 5 6 7 6 9 IO VIRUS DOSE LT-Ag mducmg units/cell

k-r+

52 57 j

FIG. 2. BHKPl cell killing by Ad12. Clone survivals from 8 different experiments are plotted as a function of the input virus dose expressed in terms of the multiplicity of virus required to induce T Ag synthesis in 63% of the cells [ = 1 T Ag inducing unit (TAU) per cell]. The dotted line is the slope expected if 1 TAU were equivalent to 1 cell-killing hit.

infecting unit for adult normal hamster cells and for adenovirus-induced hamster tumor cells when measured by immunoflourescence staining for Ad2 structural antigens (Strohl et al., 1966). Values for T Ag-producing cells in excess of 80% could not be determined accurately due to detachment of someof the infected cells and to the presenceof scattered fluorescing material. Cell survival decreased with increasing virus dose, but in a manner which indicated that the cells were killed less efficiently as the moi was increased beyond that required to yield specific fluorescence in 80-100% of the cells. The interesting feature of these curves, however, was the fact that a multiplicity of 10 PFU per cell emerged as the virus dose equivalent to 1 killing hit.2 The 2 One cell killing hit is here defined, from the Oorder term of the Poisson distribution, by the dose of virus which reduces the clone survival to 3770 of the control value.

BY Ad12

645

similarity between this value and the TAU of 14 PFU per cell suggested that the two phenomena may be related, i.e., that cells induced to synthesize T Ag were those which ultimately died. This hypothesis was tested by combining data from several experiments in a semilogarithmic plot of clone survival vs. virus dose as shown in Fig. 2. Since the relationship between T Ag frequency and cell killing was being tested, the virus dose was calculated for each experiment in terms of multiples of the virus input equivalent to one TAU as defined above. The dashed line in Fig. 2 was drawn as a one-hit curve with the slope expected if one TAU were equivalent to one cell-killing unit. Several points may be noted: (1) The experimental values were consistent with a one-hit curve, indicating that one infecting virus particle was sufficient for cell killing. (2) The experimental points actually lay somewhat below the theoretical curve as drawn, at a position indicating that one cell killing unit was equivalent to approximately 0.5 TAU, i.e., that cell killing or its demonstration was more efficient than the induction of detectable T Ag. (3) As already suggestedby Fig. 1, the fraction of cells surviving always reached a plateau level (510% of the infected cells) as the virus dose approached that required to achieve a 100% T Ag response.Further large increases in virus input (up to NO-fold) reduced the clone survival only slightly. The killing of hamster cells by Ad12 thus appeared to be one of the possible outcomes of infection initiated by single Ad12 particles rather than a nonspecific “toxic” response due to massive quantities of infected cell homogenate. Cell killing as a viral genme junction. The question of whether killing resulted from a function of the Ad12 genome was investigated by two kinds of experiments. First, the cell-killing titer and T Ag-inducing titer were assayed as before, with an Ad12 preparation that had been purified by banding in a CsCl density gradient. If the major killing activity resided in something other than intact virions (e.g., free capsid subunits, Levine and Ginsberg, 1968), the ratio of killing titer to T Ag-inducing titer should be greatly reduced in a purified preparation. That this was not the case may be seen in

646

STROHL

Fig. 2, where the result obtained with the gradient-purified virus is represented by the open triangles. Second, the sensitivity to inactivation by ultraviolet (UV) irradiation of the cell killing function was compared with that of viral infectivity and ability to initiate T Ag synthesis. After irradiation for various times as described in Methods, the virus samples were assayed as follows: (1) Residual infectivity was measured as the plaque-forming titer in the standard HEK cell assay system. Survival was expressed as a fraction of the unirradiated virus titer. (2) Survival of the T Ag synthesizing function wax determined by infecting BHK21 cells grown on coverslips with serial dilutions of a given sample of unirradiated or irradiated virus. Calculations of surviving T Ag-inducing particles were based upon the frequency of T Ag positive cells in samples infected with dilutions which gave lessthan 20 % positive cells. In this way complications due to multiple infection, which would lead to a multihit survival curve, were avoided. Survival after irradiation was expressedas a fraction of the titer of the unirradiated material. (3) The loss of cell-killing ability was assayed by comparing clone survival after infection with virus irradiated for 0, 0.5, and 2 min. By varying the input dose of irradiated virus, the relative amount of virus required to give an average of 1 cell-killing hit per cell (i.e., the dose giving a clone survival of 37 %) could be calculated and this translated into the titer of surviving cell killing particles per ml in the undiluted material. The fraction of killing virus surviving the irradiation was then given as the killing titer in the irradiated sample divided by the killing titer of the unirradiated sample. Results of these three determinations made in one such experiment are shown in Fig. 3. As reported by others (Carp and Gilden, 1965; Gilead and Ginsberg, 1966), initiation of T Ag synthesis was somewhat less sensitive to UV irradiation than was plaque formation (a 2-fold difference in the rates of inactivation). Both were single-hit events. The UV sensitivity of the cell killing ability of Ad12 was quite similar to that of T Ag induction and plaque formation, even

U.V DOSE (MINUTES1

FIG. 3. Inactivation of infectivity, T Ag inducing capacity, and cell-killing capacity of Ad12 by ultraviolet irradiation. Irradiation conditionsare given under Materials and Methods. Infectivity wasmeasuredby plaqueassay,T Ag by immunofluorescence,and cell killing by clone survival. Details of eachcalculation aregiven in the text.

though a precise estimate of the slope could not be made from the data available. These findings are clearly compatible with the view that cell killing requires some functional activity of the viral genome. The Surviving Cell Fraction The experiments thus far described were done with cell cultures that were in the exponential growth phase at the time of infection and were plated under conditions favoring multiplication after the adsorption period. Both Fig. 1 and Fig. 2 demonstrate that under these conditions the efficiency of cell killing diminished greatly when the input dose of virus was increased beyond about 4 TAU per cell (which by the Poisson distribution is the dose at which 98% of the cells should be infected). The “persistent” survivors represented 5-10% of the infected population. Is survival a genetically stable cdl function? The possibility that the surviving cells were genetically more resistant to Ad12 infection

BHK

CELL

KILLING

than the majority of the initial cell population was tested by reinfection of the surviving cells 24 hours after the primary infection. Cultures containing 6.5 X lo5 logarithmically multiplying cells were infected with 11 TAU of Ad12 per cell. Uninfected controls and infected cells were plated immediately after the adsorption period to determine the clone survival by the ordinary method, while replicate cultures were fed with 10% FC medium to permit continued multiplication. After 24 hours, cell survival in a control culture and in an infected culture was again determined by cloning. In addition, one infected culture was reinfected with 10 TAU per cell, and following the adsorption period, these reinfected cells were also plated to determine survival of cloning ability. Cell counts indicated that both infected and control cultures had increased 1.8-fold in number during the 24 hours following the primary infection. The clone survival data are shown in Table 1. The survival of infected cells plated at 0 time was 7.7 %, i.e., of the expected magnitude. At 24 hours the surviving cell fraction had decreased to 1.2 % of that found for the control cells. After reinfection this surviving fraction was further reduced to 0.18% of the control cells. Expressed in terms of those cells surviving the primary infection, approximately 15 % survived the reinfection. Thus the surviving population was about as susceptible to infection and killing as was the original population of cells. Cell division and survival. Various incidental observations suggested that the survivors might represent those cells that TABLE SUSCEPTIBILITY

OF CELLS

SURVIVING

An12

BY

were able to divide after the infection but before the killing event had occurred. If this were correct, it should be possible to show that cloning conditions favorable for early cell division after infection would also favor survival and, conversely, that inhibition of cell division would enhance the killing effect. One approach to this question was stimulated by experiments which indicated (a) that single uninfected BHK21 cells in cloning medium were capable of dividing within the first 24 hours after plating, and (b) that cell division was initiated more rapidly as the number of cells seeded per plate was increased. On the assumption that a feeder layer of X-irradiated cells might play a role analogous to high cell density (Fisher and Puck, 1956), the following experiment was carried out. Cultures of multiplying BHK21 cells were infected with from 0.4 to 48 TAU of Ad12 per cell and suspended with Viokase immediately after the adsorption period; serial dilutions of the infected cells were seeded either into plates containing medium only or into plates that had been seeded 24 hours previously with 5 X lo4 X-irradiated BHK21 cells. The resulting clones were fixed and stained after 5 days and counted. The results of this experiment, plotted along with data from two similar experiments, are shown in Fig. 4. The cloning efficiency of the uninfected cells was 87 % with, and 15 % without, feeder cells. The survival of infected cells was clearly influenced by the presence of feeder cells, the final persistent fraction being about 5-fold greater than that of cells cloned without feeder cells. The result was thus consistent with the hypothe1 INFECTION

Uninfected Cloned

Cloning Infected Us&l

Efficiency

a Logarithmically tion of clone survival tinued multiplication, the primary infection.

3.0

at 0 hour

x

10-i

TO REINFECTION Infected

Cloned

2.0

at 24 hours

x -

10-l

647

Ad12

Cloned

at 0 hour

WITH

at 0 time Cloned

at 24 hours

A~12 Reinfected at 24 hours, cloned 2 hours later

2.3

X

1OV

2.3

X

1O-3

3.6

X

1OP

7.7

x

10-Z

1.2

x

10-Z

1.8

x

10-s

growing cells were infected with 11 TAU either immediately after the adsorption or immediately after reinfection, with

of Ad12 per cell, and plated for determinaperiod (= 0 hour), after 24 hours of con10 TAU Ad12 per cell, of the cells surviving

STROHL

ability. Feeder cell layers were used in this case in order to achieve the maximum possible cloning efficiency. The remaining cultures were washed twice to remove unadsorbed virus and fed with medium containing 0.2 % FC. One culture of each set was assayed for clone survival at 24 hours after + Feeders 0----N--e infection, while one additional control and one culture infected with 2.7 TAU per cell were assayed for clone survival at 48 hours after infection. Cell counts were done in the presence of trypan blue to distinguish the viable cells. Essentially all cells were viable by this criterion. As expected, the number of cells in the uninfected cultures did not change over the 2-day period, nor did their cloning efficiency decreaseduring this period in low serum medium (32 %, 27 %, 56% at I 2 3 4 5 6 7 8 9 IO 48 0, 1, and 2 days, respectively). VIRUS DOSE (T-A~J inducing unlts/celi 1 The results are shown in Fig. 5, where the FIG. 4. Influence of X-irradiated feeder cells on fraction of cells retaining the ability to form the killing of BHKPl cells by Ad12. Infected and clones is plotted against the virus dose exuninfected cell suspensions were assayed for survipressed in TAU per cell. The effect of convai of clone-forming ability by seeding either in tinued maintenance of infected cells under the presence or in the absence of 5 X lo4 X-irraconditions not permitting muliplication was diated BHK21 cells. The different symbols represent independent experiments. Virus dose is ex- dramatic. The use of feeder cells accentuated the difference, as the infected cells plated at pressed as in Fig. 2. time 0 cloned with extremely high efficiency. After infection at the highest multiplicity, sis that increased opportunity for cell roughly 50 % of the cells formed clones when multiplication after infection enhanced the plated at time 0; only 0.13% retained this likelihood of cell survival. capacity 24 hours after infection (although The converse approach, that is, prevention they had not yet lost the capacity to exclude of any multiplication for a period of time trypan blue). Loss of survival did not conafter infection, took advantage of the obsertinue beyond 24 hours, as the fraction of vation that BHK21 cells could be brought cells surviving after 48 hours was identical to a Gl-arrested state, with no detectable to that surviving after 24 hours. Thus the multiplication, by maintenance for 2 days first 24 hours after infection appears to be under medium enriched with a low concencritical in determining the ultimate fate of tration (0.2 %) of FC (Strohl, 1969a, b). an infected cell. The vast majority of inPlates were seeded with 3 X lo5 cells 3 fected cells, which were killed when cell days before infection. After 24 hours, they multiplication was inhibited, gave rise to at were washed twice with HT medium conleast one descendant capable of forming a taining no serum and fed with HT medium clone when placed under conditions favoring containing 0.2 % FC. Forty-eight hours later, cell multiplication immediately after infecwhen ceh multiplication had ceased, they tion. Even under growth restricting condiwere infected with 0.15, 0.7, 2.7, or 5.9 TAU tions, the killing curve tended to flatten out of Ad12 per cell. One control culture and one each infected with 2.7 and 5.9 TAU were at higher multiplicities, suggesting a persuspended with Viokase immediately after sistent surviving fraction of between 10” and the adsorption period, and serial dilutions 10-4. High multiplicity infection usually rewere plated onto X-irradiated feeder cells for determination of survival of cloning sulted in some detachment of the infected

\

0 L

BHK

CELL

KILLING

-I

I

2 3 4 5 VIRUS DOSE ITAWCELLI

6

FIG. 5. Effect of cell multiplication, after Ad12 infection, on cell survival. Non-multiplying BHK21 cells were infected as described in the text and assayed for clone survival in the presence of X-irradiated feeder cells either immediately following the adsorption period (O ), 24 hours later (O), or 48 hours later (0 ). Virus dose is expressed as in Fig. 2.

celIs from the culture dishes by 2448 hours after infection. These detached cells would not be recovered in the usual cloning procedure, and therefore would be discarded when cloning was done at 24 hours but would have had the opportunity to reattach when cloning was done at 0 time. Consequently, to test whether the preceding results could have been affected by these detached cells, the cells found floating at 24 and 48 hours after infection were plated onto feeder cell layers to determine whether they could give rise to colonies. No colonies were ever recovered from such cells. Origin of mviwing clone formers. These findings raised a question of fundamental importance in relation to the process of transformation: did those cells which survived and which ultimately gave rise to transformed progeny (Strohl, 1969a) synthesize T Ag during the first 2448 hours after infection, or did they escape this expression of the viral genome as well as the

BY Ad12

649

killing function? The following experiment demonstrated that cells registering as T Ag (+) could give rise to clones, but did so by producing daughter cells which were T Ag (-). Cells were infected with Ad12 at a moi of 1.4 TAU per cell. Immediately after adsorption the monolayers were suspended with Viokase. Dilutions of the suspended cells were plated in cloning medium onto coverslips at cell densities low enough to permit the progeny of individual cells to be distinguished. The resulting clones were fixed, stained, and examined for T Ag immunofluorescence at 1, 2, and 3 days after infection. Standard assays gave a figure of 10% for surviving clone formers among the suspended cells. The figure for moi of 1.4 TAU was obtained from the observation that 76 % of the cells present at 0 time ultimately synthesized T Ag in monolayer cultures. Use of the Poisson expression then indicates that 34% would be infected with 1 TAU, and 42% with 2 or more TAU. The observations can be summarized as follows. (1) Of 243 cells examined at 1 day after infection, 31% (75 cells) were T Ag (+). They were distributed in 61 clonal groups, such that 52 were single T Ag (+) cells, 7 were doublets, 1 was a triplet, and 1 was a group of 6 cells. Thus, approximately 16 % of the attached T Ag (+) cells divided at least once in the first 24 hours, and both daughter cells contained T Ag. (2) By 2 days, most of the clones contained only T Ag (-) cells. Eleven clones, 2 to 19 cells in size, were found to contain a single T Ag (+) cell each, and in addition 7 single T Ag (+) cells not associated with clones were seen. By 3 days there were even fewer single T Ag (+) cells, all of which were found associated with clones consisting of negative cells. Although these observations permit no definitive quantitative interpretation, limited conclusions seem warranted: (1) Early after infection, division of successfully infected cells did occur. (2) While the capacity to synthesize T Ag was at first distributed to daughter cells for 1 or 2 divisions of a few infected cells, this probably represented the distribution of input virus genomes, or viral gene products, rather than replication of the

650

STROHL

genome, since all clones thereafter contained no more than one T Ag (+) cell. (3) The finding that most of the T Ag (+) cells were single and that their number decreased progressively was consistent with the data indicating that early production of T Ag is correlated with cell death. Presumably T Ag (+) cells were lost progressively due to detachment from the coverslip. (4) Conversely, the cells comprising the successfully growing clones were free of T Ag. These probably arose in either of two ways: (a) from cells in which T Ag was never synthesized and (b) from T Ag (-) descendants of T Ag (+) cells. The latter probably account for the majority of the survivors when cell multiplication is promoted, while the former could account for the very small fraction of survivors found when multiplication is prohibited for 24 hours (cf. Fig. 5). DISCUSSION

Killing of BHK21 cells abortively infected with Ad12 has been shown to be the result of viral genome expression and to be successfully accomplished by one functional virus particle. In these studies, the synthesis of T Ag has been used as the indicator of viral genome expression. When cell multiplication is restricted for 24 to 48 hours after infection, the fraction of cells killed is very similar to the fraction of cells synthesizing T Ag (as well as to the fraction of cells stimulated to begin DNA synthesis, see Strohl, 1969b) implying that both responses to infection occur in the same cell. This in no way implies that T Ag itself kills the cell, but rather that any cell in which an Ad12 genome has successfully initiated transcription of mRNA is likely to be killed. Since Ad12 DNA synthesis in hamster cells is undetectable (Doerfler, 1969), and structural antigens are not synthesized in detectable amounts (Pope and Rowe, 1964), it seems unlikely that the killing results from newly synthesized fiber protein (Levine and Ginsberg, 1968). The frequency of hamster cell metaphase figures with some chromosomal aberrations has previously been correlated with the frequency of killing of multiplying cells infected with Ad12 (zur Hausen, 1968b). In

the accompanying paper (Strohl, 1969b), Ad12 infection of Gl-arrested (nonmultiplying) BHK21 cells is shown to induce cellular DNA synthesis which is followed at 22 hours after infection by mitoses containing only occasional chromatid breaks. At times later than 27 hours after infection, mitoses consisting of extensively fragmented or “pulverized” chromatin are seen to accumulate. Such cells would obviously not be viable, but those with only one or two chromatid breaks might be. It is conceivable that the endonuclease found associated with adenovirus virions could produce this damage to the cell genome (Burlingham and Doerfler, 1969), but it is not yet known whether it is synthesized during the Ad12 abortive infection of hamster cells. That this observed range of severity of chromosomal damage is probably influenced by the physiological state of the cells during the 24-hour period following infection is suggested by the observations regarding the great influence of this period upon cell survival. The proportion of cells escaping irreversible damage was shown to depend upon whether the infected cells are placed immediately under conditions favoring exponential growth or are held in the nonmultiplying, Gl-arrested state. In the former case, 10% to 50% of the infected cells yield clones (Figs. 4 and 5) while in the latter case as few as 0.13 % were found to have retained this capacity (Fig. 5). One view of this might be that rapid multiplication after infection simply dilutes out the active genomes among the progeny cells to the point where one cell receives no genome and gives rise to a colony. Such a description could explain the origin of the mixed colonies in the experiment described in the last experimental section, since 34% of those cells received only 1 TAU. However, while this might occur after infection with a few TAU per cell, a random distribution of genomes clearly could not explain the survival of cells after infection with more than 10 or 20 TAU per cell. This suggests such possibilities as (1) asymmetric distribution of virus genomes to daughter cells, such that one daughter receives a “unit” of all the genomes, and the other receives none, (2)

BHK CELL KILLING repression or degradation of additional genomes by the activity of the first genome which begins transcription, or (3) repression or degradation of viral genomes during some particular phase of the cell division cycle. Degradation of infecting Ad12 genomes in logarithmically multiplying hamster cells has recently been observed (zur Hausen and Sokol, personal communication), but evidence on the other points is currently lacking. In any case, the infection of a randomly growing cell population must lead to a complex mixture resulting from the interplay of such variables as (1) cells infected at various stages of the division cycle, (2) cells growing with somewhat different generation times, (3) virus particles being uncoated and initiating transcription at various times after infection. In somecells an entire division cycle, or even two, might be accomplished before virus genomefunction begins, while in others, transcription leading to cell killing might occur before one cell division is completed. The exact coincidence of one phase of the cell cycle with the synthesis of one specific Ad12 gene product may lead to cell death, while the same Ad12 function may have no effect at some other phase of the division cycle. Experiments of the type done with productively infected synchronized cell cultures (Hodge and Scharff, 1969) would be necessary to explore this possibility. This raises interesting questions with respect to neoplastic transformation in the BHK21 cell system. In another report (Strohl, 1969a), it was shown that someclone formers, presumably T Ag (-), carry and transmit to daughter cells the information for eventual transformation. Are these cells in which genome expression is delayed for several days after infection, allowing time for random damagesto inactivate the cell killing gene(s)?What is the state of the viral genome in these surviving cells (zur Hausen, 1968a; Doerfler, 1968)? Doerfler concluded from his results showing physical association between the infecting virus genome and the genome of multiplying BHK21 cells, that the viral DNA was replicated along with the cell DNA. The present results on killing pose the additional possibility that the cells in which

BY Ad12

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the Ad12 DNA is incorporated and transcribed shortly after infection are the cells that ultimately die. The further study of events following infection in the relatively simple Gl-arrested cell system, and the use of synchronized cell cultures, may provide further insight into the much more complex logarithmically growing cell system. The accompanying paper (Strohl, 1969b) discusses the effect of Ad12 infection on DNA synthesis in Gl-arrested cells and its relation to cell killing and T Ag synthesis. ACKNOWLEDGMENTS The author gratefully acknowledges

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