Regulation of 2′5′-oligo(A) polymerase activity in quiescent human fibroblasts treated with interferon

VIROLOGY

111,

666-670

(1981)

Regulation of 2’5’-Oligo(A)

INDIRA

Department

of

Biological

Polymerase Activity in Quiescent Treated with Interferon’ KRISHNAN

AND

Sciences, State University

CORRADO of

Accepted February

Human Fibroblasts

BAGLION?

New York at Albany, Albany, New York 12222 24, 1981

Treatment of human foreskin fibroblast cultures with interferon results in a dosedependent increase in 2’,5’-oligo(A) polymerase activity and inhibition of encephalomyocarditis virus RNA synthesis. Confluent fibroblasts continuously treated with 100 units/ ml of interferon show an increase in 2’,5’-oligo(A) polymerase activity for the first 24 hr and a subsequent decrease in activity that is correlated with an attenuation of the antiviral state. Possible reasons for this decrease in 2’,5’-oligo(A) polymerase activity have been investigated. No changes in the growth characteristics of the quiescent fibroblasts have been detected upon treatment with interferon. Enzymatic activities that degrade 2’,5’-oligo(A) are not increased upon prolonged treatment with interferon. We conclude that the 2’,5’-oligo(A) polymerase activity in quiescent fibroblasts is unstable and is possibly subject to regulatory mechanisms, which prevent maintenance of high levels of this enzymatic activity.

Interferon induces the synthesis of a group of proteins that can be detected by gel electrophoresis and autoradiography (1, 2). Synthesis of these proteins begins few hours after addition of interferon to cell cultures, peaks at different time intervals and declines afterward (3). The induction is apparently regulated at the transcriptional level and may be transient (3). Cells treated with interferon have elevated levels of at least two enzymatic activities, a protein kinase and an oligoadenylate polymerase or synthetase (for references see Ref. (4)). Both enzymes are activated by double-stranded RNA and the polymerase can be accurately quantitated in cell extracts by measuring the formation of 2’,5’-oligo(A) (5). The increase in polymerase activity in interferon-treated cells is correlated in many cases with the inhibition of replication of different viruses (6-8). To study the regulation of 2’,5’-oligo(A) polymerase activity, we have i This research was supported by Grant AI-16076 from the National Institute of Allergy and Infectious Diseases. We thank Dr. Albert J. T. Millis for providing us with cultures of fibroblasts established in his laboratory. * To whom reprint requests should be addressed. 666 0042-6822&l/080666-05$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

treated quiescent human fibroblasts with interferon (HuIFN-B) and measured synthesis of 2’,5’-oligo(A) in cell extracts prepared at different time intervals. We find that the polymerase activity initially increases, as previously reported for other cells (4), but that upon prolonged treatment with interferon this enzymatic activity decreases and that the inhibition of viral RNA synthesis declines in cells infected with encephalomyocarditis virus (EMCV). To measure the increase in polymerase activity with interferon concentration, confluent cultures of human foreskin fibroblasts were treated for 24 hr with increasing amounts of HuIFN-P. Cytoplasmic extracts were then prepared and assayed for 2’,5’-oligo(A) synthesis (Fig. 1). Extracts from control untreated fibroblasts showed a low basal level of activity; synthesis of 2’,5’-oligo(A) increased asymptotically with the interferon concentration (Fig. 1). In parallel cultures seeded in multiwell plates, the antiviral state was monitored by infecting control or interferon-treated cells with EMCV and measuring viral RNA synthesis in the presence of actinomycin D (Fig. 1). Viral RNA synthesis peaked at about 3 hr in

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i 0

I xl

40 HuIFN-p

I SO

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I I 160

(units/ml)

FIG. 1. Synthesis of EMCV RNA (0) in human fibroblasts treated with different doses of interferon and synthesis of 2’,5’-oligo(A) in cell extracts (A). Fibroblasts were grown in medium 199 with 20% fetal calf serum and seeded in 75-cm2 flasks or in multiwell plates (24 X 1.75 cm*). The cultures were confluent after 7 to 8 days and were treated for 24 hr with fibroblast interferon (HuIFN-8, 3 X 10 units/ mg, provided by the Interferon Working Group, National Cancer Institute, NIH). To prepare cell extracts, the fibroblasts from one flask were trypsinized, resuspended in medium plus serum, washed with saline, and homogenized as described (5). Assays for 2’,5’-oligo(A) synthesis contained in a final volume of 25 ph20 ~1 of cell extract, 5 mM [3H]ATP (0.2 pCi and 125 nmol per assay), 25 mAf Mg(OAc)p, 20 rg/ml of poly(I).poly(C), and the other components previously described (5). Reactions incubated for 1 hr at 30° were analyzed by chromatography on DEAE-cellulose as described (5). The amount of 2’,5’oligo(A) synthesized is expressed as nmol/60 min/ Azso unit of cell extract. To measure synthesis of EMCV RNA, control and interferon-treated cells were infected at m.o.i. 100 in serum-free medium. After 60 min additional medium and 5% fetal calf serum were added and the infection was allowed to proceed for 3 hr. At this time 5 &ml of actinomycin D was added, followed after 20 min by 10 &i/ml of [3H]uridine. After 50 min the medium was removed and the cells were resuspended in 0.5% sodium-dodecyl sulfate; 10% trichloroacetic acid was added and the precipitate was collected on glass fiber filters for counting.

with extract of interferon-treated HeLa cells (5), namely chromatography on DEAE-cellulose columns and polyethyleneimine thin-layer plates. The major product synthesized with fibroblast extract was trimer, with lesser amounts of tetramer and pentamer also formed (data not shown). These oligonucleotides activated an endonuclease, as determined by the assay previously described (9). The time course of increase in 2’,5’oligo(A) polymerase activity was next investigated (Fig. 2). Confluent fibroblast monolayers were treated with 100 units/ ml of HuIFN-/3 for increasing times and extracts prepared from these cells were assayed for 2’,5’-oligo(A) synthesis. The polymerase activity increased progressively up to 24 hr after interferon addition, but it declined afterward (Fig. 2). This decrease in 2’,5’-oligo(A) synthesis was correlated with a decline in the antiviral state, since synthesis of EMCV RNA was more inhibited in cells treated for 24 hr with interferon than in cells treated for 4 days (Fig. 2). This was established by infecting fibroblast cultures with EMCV at m.o.i. 1 to 100. At the lowest m.o.i. tested synthesis of viral RNA was inhibited in all the cultures, whereas at the highest m.o.i. synthesis of EMCV RNA was only

Days

both control and interferon-treated cells (data not shown). The inhibition of EMCV RNA synthesis was correlated with the 2’,5’-oligo(A) synthetic activity (Fig. 1). The oligonucleotides synthesized with fibroblast extracts were identified with 2’,5’-oligo(A) by the same assays previously described for the product obtained

FIG. broblasts HuIFN-6 tracts treated extracts; infected in Fig. atively

2. Synthesis of EMCV RNA (0) in human ficultured in the presence of 100 units/ml of and synthesis of 2’,5’-oligo(A) in cell ex(A). Confluent cultures in 75-cm2 flasks were with interferon and harvested to prepare cell parallel cultures in multiwell plates were with EMCV at different m.o.i. as described 1. Synthesis of EMCV RNA is expressed relto that of control untreated cells.

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marginally inhibited in cells treated with interferon longer than 3 days (Fig. 2). At intermediate m.o.i., synthesis of EMCV RNA was observed in cells treated with interferon for 3 or 4 days. The best protection against EMCV infection was therefore observed in the cells with the highest 2’,5’-oligo(A) polymerase activity. Some possible reasons for the decrease in 2’,5’-oligo(A) polymerase activity in cells continuously cultured in the presence of interferon were investigated in the following experiments. The growth characteristics of the cells were first examined. Confluent cultures in multiwell plates were treated with interferon as described in Table 1. At daily intervals the culture medium was removed and centrifuged to pellet cells released from the monolayers. Both these cells and the attached cells were counted in a Coulter counter as indicated in Table 1. No significant change in cell number or in the level of cells released from the monolayers was detected. The proportion of dead cells, as determined by trypan blue staining, was small and remained constant throughout the experiment (Table 1). The interferon con-

centration in the culture medium also did not change appreciably. This was established by treating human fibroblasts with serial dilutions of the cultures supernatant. After infection with EMCV, the interferon titer was determined by the inhibition of the cytopathic effect (10). Since no significant change in this titer was detected (data not shown), it seems unlikely that the decrease of 2’,5’-oligo(A) synthesis in extracts of fibroblasts treated continuously with interferon is due to the disappearance of interferon from the medium. A decreased synthesis of 2’,5’-oligo(A) in cell extracts could possibly be caused by an increased level of enzymatic activities that degrade these oligonucleotides. To rule out this possibility, we measured degradation of 2’,5’-oligo(A) under conditions similar to those of the assay for its synthesis (Table 1). Degradation of 2’,5’oligo(A) was readily detected in all cell extracts, but it was not enhanced in extracts of fibroblasts treated with interferon for 3 or 4 days (Table 1). This assay was carried out with different input of cell extract and for different times (data not shown). The substrate, 3H-labeled 2’,5’-

TABLE

1

EFFECTS OF INTERFERONTREATMENTON FIBROBLASTCULTURES,~',~'-OLIGO(A)DEGRADATIVE CELLEXTRACTS AND 2',5'-OLIGO(A)POLYMERASE IN CULTURE MEDIUM"

Cell Days of treatment

Control

number

(X10e4) Interferon

0 1

9.4 9.8

9.0 9.6

2 3 4

10.0

9.4

10.0

10.0

9.0

9.0

Cells in supernatant (% of cell number) Control

Interferon

0.4 0.3 0.3 0.3 0.4

0.4 0.5 0.4 0.3 0.4

Degradation 2’,5’-oligo(A) (% degraded) 68 81 77 52 51

of

ACTIVITY IN

Synthesis oligo(A) Expt

0.1

1

of 2’5’(nmol) Expt

0.5 0.7 2.4

0.2 0.6 0.6 1.3

1.8

1.6

2

a Confluent cultures of human fibroblasts in multiwell plates were treated with 100 units/ml of HuIFN8. Cell number per well was measured by counting trypsinized cells in a Coulter counter. The cells present in the supernatant medium were counted after centrifugation and resuspension. Viable cells in each sample were measured by Trypan blue staining; more than 99% of the cells were viable. Degradation of 2’,5’-oligo(A) by extracts of cells cultured in 75-cm* flasks was assayed as previously described (5) by incubating 1 fl 2’,5’-[3H]dligo(A) for 30 min in a ‘757~1 assay containing 60 ~1 of the cell extract and the other components described for’the assay of 2’,5’-oligo(A) synthesis (see Fig. l), with the exception of poly(I).poly(C) and labeled ATP. Synthesis of 2’,5’-oligo(A) with tissue culture medium was assayed as described in Fig. 1 for cell extract. The nanomoles synthesized in 60 min in a 25-~1 incubation containing 10 ~1 of culture medium are indicated. A different fibroblast strain was used in each experiment.

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oligo(A), was added at concentrations close to those found in interferon-treated L cells infected with EMCV (11). Under these conditions, the kinetics of 2’,5’-oligo(A) degradation was characteristic of substratelimited enzymatic reactions. Under the conditions chosen to study 2’,5’-oligo(A) degradation in extracts of interferontreated cells, about 50-80% of the substrate is degraded (Table 1). This degradation is enhanced only in cells treated for 24 or 48 hr, in agreement with the finding of Kimchi et al. (8) who reported an increased 2’-phosphodiesterase activity in interferon-treated cells. The recent observations that 2’,5’oligo(A) polymerase activity is present in the serum of mice treated with interferon (1.2) and that this enzymatic activity is found in two different membrane-coated RNA viruses released from infected cells (IS), prompted us to assay the culture medium obtained from interferon-treated fibroblasts for 2’,5’-oligo(A) synthesis (Table 1). A significant activity was detected in the medium obtained from cells treated for 3 or 4 days. This enzymatic activity could be adsorbed onto columns of poly(1). poly(C)-agarose (4). When 1 ml of medium was passed through such column, in the subsequent incubation under the conditions previously described (6) about 6% of the substrate [3H]ATP was converted into 2’,5’-oligo( A). These oligonucleotides were analyzed by DEAE-cellulose chromatography and found to be predominantly trimer and tetramer (data not shown). The oligonucleotides also activated an endonuclease in the assay described (9). In the experiments reported above we observed a decrease in 2’,5’-oligo(A) polymerase activity in extracts of human fibroblasts continuously cultured in the presence of interferon, and an increase in the synthesis of viral RNA in these cells infected with EMCV. It is possible that the level of other interferon-induced enzymatic activities follows a similar pattern of induction and decline, though this remains to be established. A decrease in the level of such enzymatic activities might also be relevant for the increased EMCV RNA synthesis.

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The regulatory mechanism responsible for the decrease in 2’,5’-oligo(A) polymerase activity after prolonged treatment with interferon is unknown. A decrease in this enzymatic activity was observed in HeLa cells treated with interferon, washed, and cultured in interferon-free medium (5). This decrease could be accounted for by the increase in cell mass, by postulating that synthesis of 2’,5’-oligo(A) polymerase ceased upon removal of interferon and that the enzyme is diluted out by cell growth. In HeLa cells the polymerase appears to be stable (5). These observations cannot explain our present findings. The fibroblasts do not multiply and the interferon titer in the medium does not change appreciably. This agrees with the finding that no detectable interferon is removed from medium repeatedly used to treat cell cultures (15). Another level of regulation of interferon-induced proteins has recently been described (3). The synthesis of these proteins ceases after a characteristic time interval from the addition of interferon. The synthesis of interferon-induced proteins may thus be transient, in agreement with the observation that after an initial increase the 2’,5’-oligo(A) polymerase activity levels off in cells treated with interferon (5) and that addition of antiinterferon antibodies prevents further increase of this enzymatic activity (16). These regulatory mechanisms, however, cannot account for the present finding that the polymerase activity actually declines in fibroblasts continually exposed to interferon for days. A plausible explanation for this finding is that the polymerase is unstable in fibroblasts. Some 2’,5’-oligo(A) polymerase activity appears in the medium of fibroblasts treated with interferon at the same time that this enzymatic activity decreases in the cells. This was repeatedly observed in different cultures of independently established fibroblast lines (Table 1 and data not shown). No significant polymerase activity was detected in the medium of untreated fibroblasts. The 2’,5’-oligo(A) polymerase does not appear to be released from broken cells, since no significant

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fraction of dead cells was found in detached cells or in monolayers. Therefore, the level of enzymatic activity detected in the medium cannot be accounted for by the lysis of dead cells. We cannot say at this time whether the enzymatic activity found in the medium is equivalent to that lost by the cells, because the assay for 2’,5’oligo(A) synthesis is carried out in the presence of different cellular and medium components. We have no explanation for the presence of 2’,5’-oligo(A) polymerase activity in the culture medium, though it is tempting to speculate that an intriguing regulatory mechanism causes the release of this enzyme from fibroblasts. REFERENCES 1. GLJPTA, S. L., RUBIN, B. Y., and HOLMES, S. L., Proc. Nat. Acad. Sci. USA 76,4817-4721(1979). 2. KNIGHT, E., JR., and KORANT, B. D., Proc. Nut. Acad. Sci. USA 76, 1824-1827 (1979). 3. RUBIN, B. Y., and GUPTA, S. L., J. ViroL 34,446454 (1980). 6. BAGLIONI, C., Cell 17,255-264 (1979).

5. MINKS, M. A., BENVIN, S., MARONEY, P. A., and BAGLIONI, C., J. BioL Chem. 254, 5058-5064 (1979). 6. BAGLIONI, C., MARONEY, P. A., and WEST, D. K., Biochemistry l&1765-1770 (1979). 7. BALL, A. L., J. ViroL 94,282-296 (1979). 8. KIMCHI, A., SHULMAN, L., SCHMIDT, A., CHERNAJOVSKY, Y., FRADIN, A., and REVEL, M., Proc. Nat. Acad. Sci. USA 76,3208-3212 (1979). 9. BAGLIONI, C., MINKS, M. A., and MARONEY, P. A., Nature (Lodon) 273,684-687 (1978). 10. BORDEN, E. C., and LEONHARDT, P. H., J. Lab. Clin. Med 89,1036-1042 (1977). 11. KNIGHT, M., CAYLEY, P. J., SILVERMAN, R. H., WRESCHNER, D. H., GILBERT, C. S., BROWN, R. E., and KERR, I. M., Nature (London) 288. 189-192 (1980). 12. KRISHNAN, I., and BAGLIONI, C., Nature (London) 285.485-488 (1980). 19. WALLACH, D., and REVEL, M., Nature (London) 287. 68-70 (1980). A. G., BROWN, R. E., and KERR, 1-b. HOVANESSIAN, I. M., Nature (London) 268.537-540 (1977). 15. BUCKLER, C. E., BARON, S., and LEVY, H., Science 152.80-82 (1966). 16. SHULMAN, L., and REVEL, M., Nature (Lo&m) 288.98-100 (1980).