Role of Interferon-Induced Enzymes in the Antiviral and Antimitogenic Effects of Interferon

ROLE OF INTERFERON-INDUCED ENZYMES IN THE ANTIVIRAL AND ANTIMITOGENIC EFFECTS OF

INTERFERON

M. Revel, A. Kimchi, L. Shulman, A. Fradin, R. Shuster,t E. Yakobson.$ Y. Chernajovsky. A. Schmidt, A. Shure,

and R. Bendori Department of Virology

Weizmann Institute of Science Rehovot, Israel

INTERFERON-INDUCED ENZYMES AND THE ANTIVIRAL STATE

Exposure of human or animal cells to their respective interferons (IFs) produces, within a few hours, an increase in several enzymes, whose function appears to be mainly related to regulation of protein biosynthesis. FIGURE 1 summarizes what is known of the mechanism of action of three such enzymes: protein kinase PK-i, (2’-5’) oligo A synthetase E, and phosphodiesterase 2’-PDi. (For a complete review and bibliography see ref. 1). Induction of these enzymes was seen in our laboratory in human fibroblasts and Namalva cells, in monkey BSC-1 cells, in bovine MDBK cells and in mouse L cells and splenic lymphocytes. Using a microassay that allows the determination of the enzyme levels with about 25,000 cells (one well of a 96-hole microplate], the kinetics of induction were determined.’A typical lag period of 2-4 hr is observed after IF addition and before the enzyme levels begin to rise. Maximum increase is reached after 18-24 hr. With actinomycin D, it was possible to show that in mouse L cells, the transcription step necessary for induction of the protein kinase and of the (2‘-5’) oligo A synthetase takes place during the first 2 hr; actinomycin D inhibits i f added during the first 2 hr. but if added later, a phenomenon of superinduction is observed.‘ TABLE 1 shows that, in contrast, cycloheximide inhibits induction even if added late after IF, indicating that translation of the mRNA transcripts continues throughout the induction period. It can also be seen from TABLE 1 that in human fibroblast FS11, the transcription step for the (2’-5’) oligo A synthetase occurs more slowly than in L cells. With anti-IF antibodies, it can be shown (FIGURE 2) that the continued presence of IF on the cell surface is required for induction. Antibodies do not penetrate the cells, and their ability to inhibit even as late as 3 hr after IF addition, indicates that IF is still outside the cell at this point. From the data in FIGURE 2, the induction could be described as a stochastic process: the longer the time allowed for IF action, the higher the level of induction. The discrepancy between the effect of anti-IF shown in FIGURE 2 and

*The research for this paper was supported by grants from the Gesellschaft fur Strahlung and Umweltforschung [Munich, FRG] and the National Council for Research and Devel- opment [Israel].

?Recipient of a Fogarty Senior International Fellowship. Present address: Department of Riochemistry. Emory University, Atlanta, Georgia.

$Present uddress: Department of Viral Oncology, ICRF. Lincoln’s Inn Fields, London, England.

459 0077-8923/80/0350-0459 $01.75/0 \c> 1980, NYAS

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Revel et a].: Role of IF-induced Enzymes 461

TABLE 1 INDUCTION OF OLIGO-ISOADENYLATE SYNTHETASE*

Mouse L929 Cells Inhibitor Added Cycloheximide Actinomycin D Actinomycin D

Human FS l l Cells

at Time = 7 0 70 7 U

-2 hr 0 hr 1 hr 1.5 hr 2 hr 3 hr 4.5 hr 0 hr No inhibitor 18hrl

2

5 2

26

-

- 100

0 20 29

100 145

133 100

-

-

- 0

17

58

132 100

-

-

-

_.. . , *Monolayer cultures of mouse or human cells were treated at time = 0, with mouse L cell

IF (200 U/ml) or with human fibroblast IF (50 U/ml). Cycloheximide (25 &ml) and actinornycin D (2 pg/ml) were added at the indicated time before or after interferon. The level of (2'-5') oligo-isoadenylate synthetase was measured in NP-40 extracts prepared 8 hr after IF. as detailed previously.' The 100% value correspond to about 15.000 cpm of [SZP]-(2'-5')ApA produced; a background of 1500 cpm for nontreated cells was subtracted.

INDUCTION OF OLIGO -1SOADENYLATE SYNTHETASE

3 0 E a.

a

X

0

1 0

In

N

c3

I

Y

1.5- Mouse L 9 2 9 Cells - - Human FS I I Cells

- - 1.0- DDED AT TIYE* 10

-I 0

0 5- Ih

0 ' " ' 1 1 1 ' " 1

0 10 20 0 10 20 TIME AFTER INTERFERON HOURS

FIGURE 2. Effect of anti-interferon antibodies added after interferon (IF) on oligo- isoadenylate synthetase E induction. Monolayer cultures of L929 or FS l l cells [about 2.5 x 10' cells per well of a 96-weH microplate) received 50 U/ml mouse L cell IF or 15 U/ml human fibroblast IF, respectively. The level of enzyme E was measured at the indicated time as described.' Antibodies to mouse L cell IF [gift from G. Galasso. NIH) or antibodies to human fibroblast IF obtained in rabbits,'' were added (in amounts sufficient to block the antiviral effects of the concentrations of IF used) at the indicated times after IF addition. The lower activity of the L cells only reflects use of less radioactive ATP in the assay.

462 Annals New York Academy of Sciences

1.Rapid binding 2.1nteraction w i t h Interferon s t i l l on 3.Nuclear 4,lnterferon-induced of interferon species specif ic the c s l l surface response e n z m s s t a r t to

receptor ( t r a n s c r i p t i o n ) accumulate ( t r a n s l a t i o n )

rapid nmbrane I n t e r n a l i z a t i o n Del ayed changes of interferon biochanical

and receptor ( 7 ) response

TIME: 0 h r . . .......................... ............ 1 hr ... ...... .......... 2 hr ........._. .. . 3 hr ............ . _ . . 12 hr

binding to binding to I n h i b i t i o n by antibodies A n t i v i r a l s t a t e 5uperinduction angl iosides surface component to interferon or to and enzme induc- of a n t i v i r a l even in the cold) coded by chr 21 receptor t i o n becomes Act 0 s t a t e and

r e r i s tant e n i r e by Act 0 1

FIGURE 3. Schematic view of the early events in interferon action. Scheme based on experimental evidence listed below the time scale.

that of actinomycin D shown in TABLE 1, is due to the superinduction phenomenon by actinomycin D. The slower induction in human FSll cells seen with actinomycin D (TABLE 1) is also seen with anti-IF (FIGURE 2). These data can be summarized as in FIGURE 3, which describes some of the early events in the action of interferon.

By two-dimension gel electrophoresis of proteins labeled with [35S] methionine in the treated cells, it has been possible to demonstrate the induction of about 5-6 proteins by i n t e r f e r ~ n ~ . ~ (see FIGURE 8, below]. It is not yet known which of these proteins corresponds to the identified enzymes.

The induction of the three enzymes in response to different doses of mouse IF on L cells correlates well with the inhibition of vesicular stomatitis virus (VSV) replication in the cells (TABLE 2). Correlation between the antiviral state and the induction of the (2'-5') oligo A synthetase were reported in chick cells by Ball4 and HeLa cells by Baglioni et al.5 Monkey BSC-1 cells infected by SV40 allows a more precise kinetic study of the correlation between IF-dependent enzyme induction and the inhibition of viral protein synthesis. In this system,' monkey IF is added to cell cultures already infected by sV40 for 24 hr. Subsequently, an inhibition of SV40 late viral protein synthesis appears, together with an inhibition of viral DNA replication and virus yield. The kinetics of protein synthesis inhibition are shown in FIGURE 4. The rate of synthesis of the viral T-antigen is

TABLE 2 INTERFERON-INDUCED ENZYMES AND ANTIVIRAL STATE IN L CELLS*

Experiment I Experiment I1 Protein

Interferon Kinase Oligo-isoadenylate Phosphodiesterase U/ml VSV-RNA PKi VSV-RNA svnthetase E 2' PDi

~~

0 100 15 100 4 25 2 24 35 12 5 72

10 6 80 5 20 86 50 0 100 2 42 100

1.250 0 100 100 250 0 66

"Results are in percent of maximum activity. Cells were treated for 8 hr with interferon. Enzyme assays are as described by Kimchi et aJ.* Measure of vesicular stomatitis virus (VSV) RNA synthesis as in Weissenbach et a1.2'

Revel et ul.: Role of IF-induced Enzymes 463

inhibited first, while inhibition of capsid proteins develops only after a 10-hr lag period. Most of the host proteins continue to be synthesized.' with the exception of host histone synthesis, which is inhibited similarly to the T-antigen (FIGURE 4). Both protein kinase PK-i and (2'-5') oligo A synthetase are induced in this system: the kinetics of induction of the latter enzyme are shown in FIGURE 4. The enzymes accumulate before viral protein synthesis inhibition. Studies with extracts prepared from IF-treated SV40-infected cells confirm the presence of a

HOURS POST INTERFERON

FIGURE 4. Kinetics of inhibition of viral function by monkey interferon given late (24 hr) after SV40 infection of BSC-I cells. The rate of synthesis of the various proteins was determined by[%]methionine pulse-labeling and gel electrophoresis analysis a s described: Viral DNA synthesis was measured by [3H]thymidine-labeling of Hirt-extract- able DNA. Inhibitions were calculated at various times after interferon addition in comparison to appropriate controls. The level of (2'-5') oligo A synthetase E was measured as described.'

dominant inhibitor of translation that can also inhibit protein synthesis when added to control cell extracts.'

MECHANISM OF PROTEIN SYNTHESIS INHIBITION IN SV40-INFECTED BSC-1 CELLS BY INTERFERON: SPECIFIC CLEAVAGE OF 28s rRNA

A detailed study of the effects of IF on the expression of SV40 genes in the infected cells showed' that the inhibition is most probably at the translational level. SV40 mRNAs still continue to be synthesized at the time when SV40 protein synthesis is maximally inhibited.' Normal amounts of SV40 VP-1 mRNA can be extracted from these cells, and it was shown, by translation in reticulocyte

464

2

1 '

0.5

0.1

Annals New York Academy of Sciences

RIBOSOFlAL RNA OF CELLS TREATED WITH :

SV 40 t

Interferon - t

t

0.05

0 2 4 6

migration, cm

28 s a

18s

a'

5s

FIGURE 5. Cleavage of 285 ribosomal RNA in interferon-treated SV4O-infected BSC-I cells. Cultures infected for 24 hr were treated with 100 U/ml monkey IF and 24 hours later, RNA was phenol-extracted from NP40 cytoplasmic extracts, heated 30 min to 5OoC with 13% glyoxal-44% DMSO and electrophoresed on a 1.5% agarose gel. A UV photograph of the gel stained with ethidium bromide is shown with RNA from untreated (Jeft) and IF-treated (right) SV40-infected cells. The size of fragments a and a' was calculated from the known ribosomal RNA bands.

Revel et al.: Role of IF-induced Enzymes 465

lysates, to be fully active.' This mRNA is, however, not in polyribosomes.e An increased methylation of internal adenine residues in SV40 RNA was. until now, the only noticeable change in mRNA structure." Degradation of the SV40 16s and 19s mRNA was not observed.

One of the methods that we used to study SV40 mRNA in these cells was agarose gel electrophoresis of total cytoplasmic RNA after heating in glyoxal- dimethylsulfoxide, followed by blotting onto diazobenzyloxymethyl (DBM) paper" and hybridization of the DBM-paper with nick-translated ["PI SV40 DNA.

Cytoplasmic RNA a t t=48 h f rom c e l l s t r e a t e d w i t h :

SV 40 - - + + a t t= 0 h

I n t e r f e r o n - + - + a t t.24 h

6 28 S ...... a = 1 . 4 ~ 10

18 S

6 ...... a ' = 0.35~ 10

FIGURE 6. Cleavage of ribosomal RNA in interferon-treated SV40-infected BSC-1 cells: Experiments are similar to that in FIGURE 5 comparing RNA from (left to right] uninfected untreated cells, uninfected IF-treated cells, infected untreated cells, infected IF-treated cells.

Even under these conditions, it was possible to show that 24 hr after IF treatment, the cytoplasm still contains the normal amount of 16s SV40 RNA. Surprisingly, however, examination of the agarose gel after ethidium bromide staining showed that the ribosomal RNA pattern from interferon-treated SV40-infected cells was altered." The major alteration, consistently observed after IF treatment of the cells, are two new bands of 1.4 and 0.35 x 10" daltons and a reduction in 28s RNA (1.75 x 10'' daltons) (FIGURE 5). The new bands are best explained as being derived


from the 28s RNA by a single cleavage located 20% away from one end. The cleavage is more apparent in RNA prepared from isolated ribosomes or from cytoplasmic extracts prepared after lysis of the cells with Nonidet (NP) P40. It is only barely visible in total cell RNA extracted with dodecylsulfate. There may be several reasons for this: either the NP40 detergent treatment is needed to detect the cleavage, or rather, nuclear RNA is uncleaved and its presence masks the partial cytoplasmic RNA cleavage. The cleavage appears related to the state of activity of the ribosomes, since after fractionation cytoplasmic ribosomes on a sucrose gradient, the cleavage was much more apparent in the 80s monosome peak than in the polysomal region (unpublished). Often additional rRNA fragments may be observed (e.g. FIGURE 6, at 1.0 x lo6 daltons). but these can

TABLE 3

AND UNINFECTED CELLS* ACTIVATION OF ( 2 ' 4 ' ) OLEO-ISOADENYLATE SYNTHETASE BY RNA FROM SV40-INFECTED

Cells Treatment I. BSC-I None

Interferon SV40 SV40 + interferon

BSC-1 SV40 + interferon

11. BSC-I SV40 Reticulocytes

RNA Total cell RNA

Cytop. RNA poly( rI):( rC) None polyA' cytop.RNA polyA+ RNA poly(rI):(rC)

Amount

(2'-5')01igo A Synthesis

com [3zP]-( 2'-5')ApA

9,000 7,800

13.600 18,100 1,500

36,100 150

8,800 145

9,600

*Where indicated, monolayer cultures of BSC-1 cells infected with SV40 for 24 hr were treated by 100 U/ml monkey IF (Yakobson et 01626) and 24 hr later, total RNA was extracted by phenol and 0.5% SDS. and precipitated with 2 M LiCI. From cytoplasmic extracts prepared with 0.5% NP40, cytoplasmic total RNA was obtained by phenol, 0.5% SDS- 5 mM EDTA. PolyA' RNA was purified on oligo(dT)-cellulose. RNAs were added to a 25 pI reaction containing purified (2'-5') oligo-isoadenylate synthetase E (3 pg) and [3ZP]-~-ATP (1 mM; 150 mCi/rnmol], incubated 20 hr, at 30°C and the amount of (2'-5')ApA cores formed measured after alkaline phosphate as in Kimchi et 01.'

appear also in extracts from control cells. It is unlikely that the specific cleavage of ribosomal RNA extracted from IF-treated cells, shown in FIGURE 5. is produced in vitro, since incubation of these extracts under Conditions of protein synthesis did not enhance the specific cleavage but produced different patterns of ribosomal RNA degradation in both control and IF-treated extracts. It appears therefore that a rather specific nuclease activity which cleaves 28s RNA at a single site is enhanced in the cytoplasm of IF-treated cells. The specific rRNA cleavage appeared only if cells had been both infected with SV40 and treated with IF (FIGURE 6), under conditions where the inhibition of viral protein synthesis is observed. The nuclease that produces this cleavage has not yet been identified with certainty. One interesting possibility is that RNase F, which is activitated by (2'-5') oligo A,"-'3 is responsible for this cleavage. Interferon

Revel et al.: Role of IF-induced Enzymes 467

induces the (2’-5’) oligo A synthetase in these cells (FIGURE 4). In addition, poly A‘ mRNA from BSC-1 cells fulfills the double-stranded RNA requirement of (2’-5’) oligo A synthetase (and protein kinase) with about 1% the efficiency of poly(rI):(rC) (TABLE 3). Some increase in this dsRNA-like activity was seen in total RNA from SV40 infected cells (TABLE 3). There was, however, no such increase in the cytoplasmic RNA. It is, nevertheless, likely that (2’-5’) oligo A is produced in these IF-treated SV40-infected BSC-1 cells. Hovanessian et all4 recently observed a similar pattern of ribosomal RNA cleavage in cells treated by (2’-5’) oligo A in the presence of calcium phosphate. This would be in line with a role of the (2’-5’) oligo A-dependent RNase F in the cleavage of ribosomal RNA; another nuclease, such as the RNase M of Herzberg et al.,’‘ which also splits rRNA, was not found to be increased after IF (Herzberg, personal communication). Interfe- ron’s action may thus resemble in some way, that of colicin E,.16 The degradation of the 28s rRNA may explain the inhibition of translation observed in vivo and in vitro. The cleavage may affect only a part of the cell’s ribosomes, if the idea of a localized activation of the (2’-5‘) oligo A-dependent RNase proposed by Bag1i0ni.l~ as a result of a localization of dsRNA sequences, is valid. In the intact cell, only certain ribosomes would be affected, explaining why a large fraction of the host protein synthesis continues. In the cell extracts, the effect might become more pronounced than it is in the intact cells. Clearly, the mechanism of rRNA cleavage remains to be further elucidated, but the importance of these experiments is that they focus our attention, for the first time, on rRNA rather than on mRNA as the target of IF action, at least for the antiviral effect of interferon on SV40.

ROLE OF (2’-5’) OLIGO A SYNTHESIS AND DEGRADATION IN THE REGULATION OF CELL MITOGENESIS

Interferon synthesis slows down the rate of cell division and the onset of DNA synthesis in a variety of cells (for review see ref. 18). It is not known whether these effects are mediated by an action on protein synthesis similar to that observed in the antiviral effect. Histone biosynthesis is inhibited very early after IF treatment in SV40-infected cells (FIGURE 4). and also in uninfected cells (TABLE 4). We have, however, found that the histone mRNA activity (assayed by translation of poly A- RNA in reticulocyte lysates) is also reduced by IF (unpublished). But another indication that common pathways operate for the antiviral and antimitogenic effects comes from the ~bservation’~ that (2’-5‘) oligo A cores (e.g., micromolar concentrations of 12’-5’)ApApA but not (3’-5’)ApApA) are able to inhibit the onset of DNA synthesis in Concanavalin A (Con A)- stimulated mouse spleen lymphocytes, in the same way as interferon. TABLE 5 shows that inhibition of DNA synthesis by (2’-5‘)ApApApA added to the intact lymphocyte cultures is largest when the oligonucleotides, are added together with Con A or 24 hours later, while addition at 48 hr produces only slight inhibition. In all cases, DNA synthesis was measured at 72 hr after Con A. Stimulation of DNA synthesis by Con A in these lymphocyte cultures starts at 48 hr and is maximal at 72 hr. The oligonucleotides have therefore to be added before the cell enters the S phase. This is also true for IF itself (TABLE 5 and ref. 20). Addition of the (2‘-5’) oligo A produces after 48 hr a partial inhibition of protein synthesis in these Con A-stimulated lymphocytes: polyacrylamide gel


TABLE 4

MONKEY KIDNEY CELLS* INHIBITION OF HISTONE SYNTHESIS BY INTERFERON IN UNINFECTED

Hours After Inhibition of ["SIMethionine Label Interferon Histones (70) Actin (% 1

7 hr 30 10 12 hr 35 3 18 hr 50 lfl

*Duplicate cultures of BSC-1 cells were treated with 200 U/ml monkey interferon," about one day before confluency. Cells were labeled for 2 hr with ["S]methionine (40 pCi/ml) and nuclear proteins were subjected to electrophoresis on 12% polyacrylamide gel in SDS. After autoradiography the intensity of the histone and actin bands were measured by scanning at 600 mp. The percent decrease in band intensity after IF is given.

electrophoresis showed that among others, histone synthesis is particularly affected, while many other proteins are unaffected (unpublished results). Inhib- ition of DNA synthesis by (2'-5')ApApA was also reported by Martin" in cultures of lymphoblastoid Daudi cells.

A further line of evidence indicating that the (2'-5') oligo A plays a role in the regulation of cell mitogenesis is derived from the observation that Con A- stimulated lymphocytes have a much higher level of the 2'-phosphodiesterase (2'-PDi) which degrades (2'-S')pppApApA into ATP and A M P 2 than do lymphocytes maintained without mitogen (TABLE 6). The high level of z'-phosphodiesterase produces an inhibition of the synthesis of (2'-5') oligo A in extracts of Con A-stimulated lymphocyte^.'^ A similar higher level of 2'-PDi was seen in fast growing BSC-1 cells as compared to confluent cultures of these monkey kidney

TABLE 5 INHIBITION OF DNA SYNTHESIS IN CON A-STIMULATED MOUSE SPLEEN LYMPHOCYTES BY

INTERFERON AND BY (2'-5') OLIGO-ISOADENYLATE*

DNA Synthesis at 72 hr after Addition to Con A- Time of ('H)Thymidine Incorporation stimulated Cultures Addition (cpm) Inhibition (%)

Experiment 1 None - 111.020 0

Interferon 48 hr 102.060 8 Experiment 2 None - 66.150 0

( 2'4')ApApApA 0 hr 17.665 74

Interferon 0 hr 13.175 88 Interferon 24 hr 66.050 41

(2'-5')ApApApA 24 hr 18,800 72 ' (2'-5')ApApApA 48 hr 56.765 15

'Mouse spleen lymphocytes (8 x 10' cells/ml) were treated with 2.5 pg/ml Con A. Interferon (200 U/ml) or (2'4')ApApApA (5 pM) was added 10 min later ( 0 time) or at various times after Con A. DNA synthesis was measured at 72 hours by a 3-hr pulse of 6 pCi/ml ['Hlthymidine (5 Ci/mmol). Cells were then harvested on glass fiber filters (Whatman GFC), washed twice with 5 ml cold PBS, twice with 5 ml5% TCA then with 95% ethanol, dried, and counted. ['Hjthymidine incorporated into the unstimulated lymphocytes (28.750 cpm) was subtracted from that incorporated into the mitogen-treated cells.


TABLE 6 CON A-INDUCED 2'-PHOSPHODIESTERASE I N MOUSE LYMPHOCYTES*

Degradation of (2'-5') Oligo A (dimer + trimerj

No Mitogen Con A

Crude extract 14 Fg 1500 8350 DEAE, 150 mM KCI 2.5 pg 0 10750

5 w3 5050 15550

Fraction PPm) kPm)

'Mouse spleen lymphocytes were cultured for 72 hr in RPMi 1640 with or without 2.5 pg/ml Con A. The cells were lysed in a buffer containing 0.5% NP40 and the cytoplasmic crude extract was fractionated on DEAE-cellulose to isolate the fraction containing the 2'-phosphodiesterase as in Schmidt et al." Degradation of a mixture of ["P]-[2'- 5')pppApApA and pppApApApA (total cpm - 30,400 cpm) was measured by the amount of AMP released in 1 hr at 30°C as described before." with the indicated amount of protein fractions.

cells (Kimchi, Shure & Revel, in preparation). The basal level of (2'-5') oligo A synthetase is also quite variable under various cell culture conditions.2'

These results suggest that a growth stimulus is accompanied by an increase in an enzyme which degrades (2'-5') oligo A and may prevent its action as an inhibitor of entry in the S phase. Interferon treatment increases the level of (2'-5') oligo A synthetase E by at least fourfold over its basal level in Con A-treated lymphocyte^.'^ This high level of (2'-5') oligo A synthetase, activated by the endogenous dsRNA-like RNA of the cell, would overcome the effect of the 2'-phosphodiesterase and produce enough accumulation of (2'-5') oligo A to block

I NTE- MITOGEN -- -

I v V A T P ( 2 l - 5 ' ) OLIGO A 2 ' -PHOSPHO- AMP

( ~ ' - ~ ' ) P P P A P A P A , , ,

t I

I RNASE F

FIGURE 7. Schematic view of the regulation of mitogenesis by (2'-5') oligo A synthetase E and 2'-phosphodiesterase 2'-PDi. The rectangle symbolizes the cell. Arrows indicate that interferon induces E and to a lesser extent 2'-PDi2. A mitogen stimulus as Concanavalin A for lymphocytes leads to an increase in 2'-PDi activity [see TABLE 6).



entry in the S phase. The stimulation of 2’-PDi observed in IF-treated L cells (ref. 2 and TABLE 2) may actually be needed to prevent the damage that (2-5’) oligo A accumulation and RNase F activation could cause to these cells. It may be speculated that cells whose rate of division is less affected by IF treatment utilize this induction of X’-PDi to balance the increase in (2’-5’) oligo A synthetase. The ratio of (2‘-5’) oligo A synthetase over 2’-phosphodiesterase may then be a really significant parameter regulating cell division (FIGURE 7). Actually, the possibility is not excluded that exogenously added (2’-5’)ApApA cores act by competitive inhibition of the 2’-phosphodiesterase, rather than being phosphorylated into the (2’-5’)pppApApA activator of RNase F. The possibility that the (2’-5’) oligo A has other effects in the cell than just activating RNase F has also not been excluded. Nevertheless, it now seems tempting to implicate the intracellular level of (2’-5’) oligo A in both antiviral and antimitogenic effect of interferon. Since, however, IF induces at least 4-5 proteins in these lymphocytes (FIGURE 8), including the dsRNA-dependent protein kinase PK-i (Kimchi and Revel, unpublished), it would not be suprising that more complicated mechanisms are at work to produce the astonishing cell regulatory functions of interferons.

ACKNOWLEDGMENTS

Gifts of (2’-5’) oligo A from Drs. Y. Lapidot and S. Rapoport are gratefully acknowledged.

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