Eur. J. Biochem. 137, 623-629 (1983) 0 FEBS 1983

Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection

Albert0 MUAOZ and Luis CARRASCO Departamento de Microbiologia, Centro de Biologia Molecular, Universidad Autbnoma, Madrid

(Received March f7/July 22, 1983) - EJB 83 0264

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN-ol (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon-treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut-off of host protein synthesis in interferon- treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut-down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut-off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon-treated cells and this early gene expression is necessary to block cellular protein synthesis.

Animal cells treated with interferon do not support the growth of a number of animal viruses [1,2]. The step in virus replication blocked in interferon-treated cells varies with the kind of virus analyzed [3]. For picornaviruses it was first observed that no viral protein synthesis takes place in L cells pretreated with MuIFN-ol [4]. Studies on mengovirus replica- tion indicated that, though no virus replication occurred in interferon-treated L cells, the blockade of host protein syn- thesis by viral infection followed kinetics similar to that in control cells not treated with interferon [5,6]. However, it was later reported that if interferon is continuously present by repeated addition to the culture medium, the cell recuperated from the inhibition of protein synthesis and re-started the synthesis of cellular proteins [7].

Interference with cellular protein synthesis in encepha- lomyocarditis virus-infected HeLa cells treated with interferon depended on the ratio of interferon concentration and the multiplicity of infection (m.0.i.) used: the higher this ratio, the better the protection of cellular translation achieved. In cells in- fected at high m.0.i. encephalomyocarditis virus, shut-off of protein synthesis took place, the cytophatic effect developed with a timing similar to the untreated cells, and finally the encephalomyocarditis-infected cells died, even if interferon was continually added to the culture medium [8].

Though poliovirus is one of the most extensively studied animal viruses [9,10], the mechanism of the blockade of

Abbreviations. PJNaCl, phosphate-buffered saline (137 mM NaCl, 2.7 mM, KC1, 8 mM Na2HP04. 7 H20, 1.5 mM KH,PO,); p.f.u., plaque-forming units ; HuIFN-a (Ly), human lymphoblastoid interferon; SDS, sodium dodecyl sulfate; m.o.i., multiplicity of infection.

poliovirus replication by treating cells with interferon has not yet been analyzed. Besides, the inhibition of protein synthesis in poliovirus-infected cells is one of the most acute known. We wanted to analyze the mechanism of inhibition of poliovirus replication by interferon, and whether the shut-off of host protein synthesis is prevented in interferon-treated cells. Our results indicate that the shut-off of translation takes place very rapidly, in spite of viral RNA and protein synthesis being very much inhibited. These results suggest that competition of viral mRNAs for cellular messages is not involved in the inhibition of protein synthesis in this system.

MATERIALS AND METHODS

Cells and virus

HeLa cells were propagated in culture flasks (Falcon Plastics) containing 6 ml Eagle's medium as modified by Dulbecco (E4D) supplemented with 10 % newborn calf serum (E4D10) and incubated at 37 "C in a 5 % CO, atmosphere. Poliovirus type I was grown on HeLa cells in Eagle's medium as modified by Dulbecco supplemented with 1 % newborn calf serum. The fraction obtained after removal of cell debris by low-speed centrifugation was used as source of virus.

Interferon

Human lymphoblastoid interferon, HuIFN-ol (Ly), 1.6 x lo6 IU/mg protein was a generous gift of Drs Finter, Fantes and Johnston, Wellcome Research Laboratories, Beckenham, UK.

624

Virus injection and measurement of protein synthesis

HeLa cells grown in 24-well Linbro plates were infected with poliovirus at the m.0.i. described in each experiment. After 1 h incubation at 37 "C, the medium was removed and 1 ml Eagle's medium as modified by Dulbecco supplemented with 2 % newborn calf serum (E4D2) was added. Time of virus addition was considered as -1 h, and zero time was taken as when the virus is removed. Incubation at 37°C was continued until the labelling period. For this purpose, 0.5 ml methionine-free E4D1 medium and 0.11 pCi [35S]methionine (1000 Ci/mmol; 5.4 mCi/ml, The Radio- chemical Centre, Amersham) were added to the cells for I-h pulse. The medium was then removed, the cells were washed with Pi/NaCl and precipitated with 5 % trichloroacetic acid. After 5 min, the trichloroacetic acid was removed and the cell monolayer washed three times with ethanol, dried under an infra-red lamp and dissolved with 250 p10.1 M NaOH plus 1 % SDS. 125 pl were counted in an Intertechnique scintillation spectrometer.

Table 1. Inhibition of poliovirus replication in HeLa cells by human lymphoblastoid interferon

Multiplicity Interferon Poliovirus Inhibition of infection treatment yield

I 3 3 3 3 3

I1 1 1 5 5

10 10 20 20 40 40

IU/ml

-

2 10 50

200

50

50

50

50

50

-

-

-

-

-

lo-* xp.f.u./ml % 2.80 1.90 32 0.63 78 0.69 97 0.03 99

2.9 0.14 95 2.0 0.16 92 2.3 0.16 93 3.1 0.16 95 3.1 0.26 92

Analysis of the proteins synthesized in virus-infected cells

HeLa cells were grown on 30 mm petri dishes and pulsed with 5.4 pCi [35S]methionine. At the end of the pulse period, the cells were washed with 1 ml Pi/NaC1 and dissolved in 200 pl 0.02 N NaOH plus 1 % SDS and 200 pl sample buffer (62.5 mM Tris pH 6.8; 2 % SDS; 0.1 M dithiothreitol; 17 % glycerol and 0.024 % bromophenol blue as indicator). Each sample was sonicated to reduce viscosity and heated to 90 "C for 5 min. 10 p1 were applied to a 15 % polyacrylamide gel and run overnight at 30 V. Fluorography of the gel was carried out with 2,5-diphenyloxazole/dimethyl sulfoxide (20 %, w/w). The dried gels were exposed using XS-5 X-ray films (Kodak).

Viral RNA synthesis

Poliovirus RNA synthesis was measured by estimating the incorporation of [5,6-3H]uridine (40 - 60 Ci/mmol, 1 mCi/ml, The Radiochemical Centre, Amersham) into tri- chloroacetic acid-precipitable material. Actinomycin D ( 5 pg/ml) was used to inhibit cellular RNA synthesis from 30 min before viral infection to the end of the labelling period. At the times after infection indicated, 1 pCi/ml of the isotope was added, and after a 1-h pulse the cells were processed as described for protein synthesis.

Measurement of 86Rb+ content

HeLa cells grown in E4D10 medium were placed in 280 p1 of a mixture of methionine-free E4D1 medium: E4D10 medium (3:l); 0.2 pCi "Rb+ (1 mCi/ml, The Radiochemical Centre, Amersham) was added, and the cells were incubated for 18 h at 37 "C. Virus infection was then carried out while maintaining constant the 86Rb+ concentration. At the times indicated, the cells were pulsed with 0.14 pCi [35S]-methionine. After 1 h incubation, the medium was removed and the cells were washed three times with 1 ml Pi/NaCl, and 0.5 ml 5 % trichloroacetic acid was added to extract the 86Rb+ from the cells. The radioactivity of 0.4 ml of the trichloroacetic acid extract was determined by estimating the Cerenkov radiation in a liquid scintillation counter. The level of protein synthesis was estimated in parallel cultures as described above.

Source of inhibitors

Source of inhibitors used : hygromycin B (Lilly Labora- tories) ; cycloheximide (Calbiochem) ; actinomycin D and guanidine hydrochloride (Sigma).

RESULTS

Inhibition of poliovirus replication in HeLa cells treated with HuIFN-a ( L y )

Production of infectious poliovirus by treatment of cells with human lymphoblastoid interferon, HuIFN-a (Ly), is shown in Table 1. A dose-dependent inhibition of poliovirus by interferon treatment of HeLa cells was observed. As few as ten units of interferon decreased production of infective poliovirus considerably and 200 IU/ml HuIFN-a (Ly) led to the production of only 1 % of control infectious poliovirus particles. This inhibition was observed over a wide range of multiplicities of infection used; even infection at a high m.0.i. (40 p.f.u./cell) produced only 8 % of poliovirus-infective par- ticles when the cells were pretreated with 50 IU/ml interferon.

The protection conferred by several interferon concentra- tions on the level of protein synthesis 24 h post-infection in HeLa cells infected at different m.0.i. is shown in Fig. 1. When a low m.0.i. was used, most cells treated with interferon survived infection, whereas at m.0.i. higher than 1 p.f.u./cell, the infected cells died (Fig. 1A) even though virus production was drastically reduced, as was shown in Table 1. When the cell confluence increased, cells were better protected and more survived (Fig. 1B). This result is in agreement with previous observations where the antiviral state was more efficiently established when cell confluence increased [l, 1 I]. We interpret these results to mean that cell survival occurs at low m.0.i. in interferon-treated cultures because only a few cells are infected initially and the subsequent cycles of virus replication are prevented, whereas if m.0.i. is high enough no protein synthesis occurs even in interferon-treated cells. To study this effect further, analysis of viral replication in a single step growth cycle was carried out.

625

A

Protein synthesis and R N A synthesis in poliovirus-infected HeLa cells pretreated with HuIFN-u (Ly )

To establish whether viral translation is inhibited in interferon-treated cells after poliovirus infection, and whether the inhibition of cellular protein synthesis takes place in interferon-treated cells, the experiment shown in Fig. 2 was done. HeLa cells were pretreated overnight with 50 IU/ml of HuIFN-u (Ly) and infected with two different multiplicities of poliovirus, 10 or 100 p.f.u./cell. Protein synthesis was esti- mated at different times from infection by measurement of total radioactive trichloroacetic acid-precipitable material, and by analysis of the individual proteins synthesized in polyacrylamide gels. In control infected cells not treated with interferon, a rapid inhibition of cellular translation took place at either m.0.i. used, followed by the exclusive synthesis of viral

B - - : 100 c

C 0 " s - 'A

'A m ._

5 5 50 m C m 0

a

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L

0 0 10 50 200LOO 0 10 50 200LOO

Interferon iU/rnl)

Fig. 1. Cell protection against poliovirus infection induced by interferon treatment of HeLa cells. Interferon was added 18 h before infection and the level of protein synthesis at 24 h postinfection was measured by a 1 h pulse of [35S]methionine. Controls were uninfected cells treated with the corresponding interferon concentration. Numbers indicate the m.0.i. used. (A) 4 x lo5 cells/l6-mm diameter well. (B) 6.5 x lo5 cells/l6-mm diameter well

iHulFN-a (Ly) 50 U/ml

Time post 1 2 3 4 5 6 7 8 9 infection (h)

polio m.0.i.

10

Control

1 2 3 L 5 6 7 8 9

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proteins. When cells were infected with 100 p.f.u./cell, viral protein synthesis started and finished earlier than when the low m.0.i. was used. In line with other experiments [6], in interferon-treated cells, shut-off occurred and was even more drastic than in untreated cultures, but no viral protein synthesis was detected at either m.0.i. studied. It seems that in cells treated with interferon, poliovirus infection triggers a suicide mechanism in which a rapid inhibition of cellular metabolic processes develops, rendering cells unable to sup- port viral replication. These results contrast with the cellular protection observed in interferon-treated cells infected under low rn.0.i. of encephalomyocarditis virus, in which cellular protein synthesis continued unabated [8]. As regards trans- lation, this observation is similar to the situation described for vaccinia virus-infected L cells grown in suspension and pretreated with mouse interferon [12,13].

The time course of viral RNA synthesis in control and interferon-treated cells under different m.0.i. is shown in Fig. 3. Although a clear inhibition of viral RNA synthesis is apparent in interferon-treated cells, there is some viral RNA synthesis. The residual RNA synthesis increases when the m.0.i. is raised. This suggests that some viral replication takes place both at the level of transcription and translation, since prior synthesis of viral polymerase should be necessary for viral RNA synthesis to take place [9,14]. However, the synthesis of viral proteins must be very low since they are not detected by analysis with polyacrylamide gel electrophoresis.

Membrane integrity in poliovirus-infected HeLa cells

Modification of membrane permeability to several com- pounds is quite general in animal viruses. These modifications are observed very early in infection when the virus penetrates the host cells, as well as late in infection when the bulk of virion proteins are synthesized [14 -161. Amongst these modifications in membrane permeability, a leakage of mono- valent cations is apparent after infection with picornaviruses _ _ [8,17-201.

P U o 2 L 6 8 io Tin h)

Fig. 2. Ejject of interferon on protein synthesis in HeLa cells infected with poliovirus. Time course and analysis of the proteins synthesized by autoradiography of the polyacrylamide gel in cells untreated (0) or treated (0) with interferon 18 h before infection. Conditions of infection were as described under Materials and Methods. Protein labelling was carried out by giving 1-h pulses with 5.4 pCi [35S]methionine at the times indicated. (A) 10 p.f.u./cell. (B) 100 p.f.u./cell. Incorporation in uninfected untreated cells was 8203 counts/min and in uninfected interferon- treated cells 8017 counts/min (zero time)

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The effect of poliovirus infection on 86Rb+ leakage both in control and in interferon-treated cells throughout infection is shown in Fig. 4. In poliovirus-infected cells, a leakage of *'Rb+ ions commenced from the third hour post infection, in such a way that most viral proteins were synthesized in cells where the ionic content had changed a lot. However, if cells were pretreated with interferon, the leakage of the membrane was delayed. Four hours after infection when cellular protein synthesis had been completely shut down, the leakage of *'Rb+ ions was almost negligible occurring from the fifth hour, which suggests that the membrane is altered progres- sively. Our previous observations indicated that for late membrane leakiness to develop, viral gene expression was necessary [21-231. This agrees with the fact that in interferon-

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Fig. 3. Inhibition of poliovirus RNA synthesis in HeLa cells by interferon. Interferon (50 IU/ml) was added 18 h before infection. Conditions of infection and blockade of cellular RNA synthesis by actinomycin D were as described under Materials and Methods. The level of viral RNA synthesis was measured by I-h pulses with 1 pCi [5,6-3H]uridine in Eagle's medium as modified by Dulbecco supple- mented with 2 % newborn calf serum. (A) 40 p.f.u./cell; (B) 10 p.f.u./cell; (C) 3 p.f.u./cell; (0 - - - 0) untreated cells; (0 - - - 0) interferon-treated

treated cells, viral gene expression is strongly inhibited (Fig. 2) and hence membrane leakiness would also be prevented.

These studies on membrane permeability alteration were widened with the hygromycin B test [24]. Protein synthesis was estimated at various times post infection in the absence or presence of hygromycin B, a non-permeant translation inhibi- tor. In agreement with previous studies on other virus-cell systems, an increased entry of this aminoglycoside antibiotic occurred late in infection in control poliovirus-infected cells, whereas, if cells were pretreated with interferon, the ap- pearance of this membrane alteration was prevented.

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Fig. 5. Effect of interferon treatment on poliovirus-induced permeability to hygromycin B. Permeability to hygromycin B of HeLa cells un- treated (0) or treated (0) with 50 IU/ml interferon 18 h before infection (10 p.f.u./cell) was estimated by measurement of the level of protein synthesis in cells pulsed with ['%]methionine for 1 h (including 1 mM hygromycin B). The controls were cells not treated with the inhibitor and infected at the indicated multiplicity. The radioactivity incorporated in uninfected cells not treated with interferon or hygromycin B was 67051 counts/min, in uninfected interferon-un- treated hygromycin-B-treated cells 65421 counts/min, in uninfected interferon-treated hygromycin-B-untreated cells 66160 counts/min and in uninfected cells treated with interferon and hygromycin B 64469 countslmin

621

t “ t I

0 2 L 6 6 1 0 0 2 L 6 8 10 Time (hl

Fig. 6 . Effect of the inhibition of viral replication on the shut-off ofprotein synthesis induced by poliovirus in HeLa cells untreated or treated with interferon. Interferon (50 IU/ml) was added 18 h before infection. The inactivation with ultraviolet light was carried out by exposing the poliovirus for 3 min to a G-E Germicidal G8T5 General Electric lamp at 4 “C at a distance of 3 cm. The guanidine (3 mM) was present where indicated, from the beginning of infection to the end of the labelling period. The level of protein synthesis was estimated as described under Materials and Methods. (A) normal virus, 5 p.f.u./cell; (B) ultraviolet-light-inactivated virus, 5 p.f.u./cell; (C) Normal-virus, 100 p.f.u./cell; (D) ultraviolet-light-inactivated virus, 100 p.f.u./cell. (0) Interferon-untreated guanidine-untreated cells; (0) interferon-treated guanidine-untreated cells; (A) interferon-untreated guanidine-treated cells; (A) interferon-treated guanidine-treated cells

Is viral gene expression necessary to block protein synthesis in interferon-treated cells?

To establish whether viral replication is needed for the inhibition of cellular protein synthesis, or whether this effect is accomplished by the input virion particles, several experiments were done. In the first set of experiments, viral replication was inhibited by ultraviolet light irradiation or by guanidine treatment (Fig. 6 ) . Ultraviolet light treatment of our polio- virus preparation decreased the infectious particles in the sample by more than two logarithmic units. Infection of control or interferon-treated cells with ultraviolet-light-inac- tivated virus clearly prevented the shut-off of host protein synthesis. Besides, the presence of 3 mM guanidine, an inhibitor of poliovirus replication, was unable to block the inhibition of translation after poliovirus infection. In the second approach, we used cycloheximide to inhibit viral translation from the beginning. Cycloheximide at a concentra- tion of 50 pM produced an inhibition of protein synthesis in cultured cells by almost 100 % [25]. At this concentration, the action of cycloheximide was reversed when the cell monolayer is washed with fresh medium. We analyzed the development of the shut-off after cycloheximide reversion, both in interferon- treated and control poliovirus-infected HeLa cells (Fig. 7). Cycloheximide was present during poliovirus adsorption and was left for three or four hours after virus removal. Release of cycloheximide inhibition resulted in a burst of protein syn- thesis. This recovery in poliovirus-infected cells after cyclo- heximide had been washed, suggested that viral protein synthesis was necessary for the shut-off to take place. The recovery did not reach 100% of control uninfected cells, perhaps for several reasons : (a) the cycloheximide reversal was not 100 %, (b) once cycloheximide was washed, the shut-off of

protein synthesis induced by viral replication took place, and (c) some viral proteins were, in fact, synthesized during the hours that cycloheximide was present, which could contribute to shut-off of cellular translation. We feel that the second reason is perhaps the one that contributes most to the initial blockade observed after cycloheximide removal.

DISCUSSION

Interferon has a number of biological effects when it comes into contact with cultured cells [l]. One of these effects is the induction of an antiviral state, that renders the cells unable to support the growth of animal viruses. The nature of this antiviral state and the exact molecular mechanism by which animal virus replication is inhibited is only partially under- stood. It is now clear that the step in the replication of different animal viruses inhibited in interferon-treated cells varies [3]. Studies on the inhibition of picornavirus growth by interferon have been mainly concerned with encephalomyocarditis virus and mengovirus, two members of the genus Cardiovirus. To our knowledge, no studies have been done with poliovirus, the best studied member in molecular terms of picornavirus belonging to the genus Enterovirus.

We described in encephalomyocarditis virus-infected cells treated with interferon two extreme situations with regard to cell survival. A suicide mechanism when high m.0.i. and low HuIFN-a (Ly) doses are used, and cell survival under low virus multiplicities and high interferon concentrations [8]. The antiviral effect of interferon on poliovirus growth seems to be based on the suicide mechanism, and no survival was ob- served even when cells were pretreated with 400 IU/ml

628

Poliovirus 135S1Met--- Poliovirus [35S1Met--- CHX CHX

Poiiovirus CHX 1 1 Poliovirus

I t t 1- -1 0 3 L 5 - 1 0 L 5 6

Time (h)

Fig. 7 . Inhibition by cycloheximide ofthe shut-off of protein synthesis induced by poliovirus in HeLa cells untreated or treated with interfiron. Cells treated ( A ) or not (A) with interferon (50 IU/ml) for 18 h were infected with poliovirus (20 p.f.u./cell) in the presence of cycloheximide (50 pM) which was removed 3 h (A) or 4 h (B) postinfection. The cells were then washed and placed in fresh medium for 30 min. The level of protein synthesis was estimated in presence of the inhibitor at the time prior to its removal and cell wash, and in its absence at the time after cycloheximide removal and cell wash. (0) Interferon-treated poliovirus-infected cells not treated with cycloheximide. (0) Interferon-untreated poliovirus- infected cells not treated with cycloheximide. CHX, cycloheximide

HuIFN-a (Ly). This reinforces the idea that poliovirus pro- duces a much more drastic interference with cellular metabolic processes than other picornavirus species.

The inhibition of host protein synthesis induced by infection of HeLa cells with low m.0.i. of encephalomyocar- ditis virus is prevented if cells are treated with interferon. This situation is not observed during poliovirus infection. Shut-off in this system is only blocked if viral gene expression is inhibited by inactivation with ultraviolet light or by the presence of 50 pM cycloheximide from the very beginning of infection. These results suggest that in interferon-treated cells some viral gene expression occurs early in infection, and this is responsible for the shut-off observed. The effect of guanidine on poliovirus replication is believed to be located mainly at the level of viral RNA synthesis, and no effect should occur on the translation of the viral RNA input. This suggests that a product ofviral translation is involved in the early inhibition of cellular protein synthesis. Whether the translation of the input viral RNA is allowed in interferon-treated cells and the replication of viral RNA is blocked, still remains to be established. If so, the inhibition of the bulk of viral protein synthesis observed late in infection in interferon-treated cells should be a consequence of the blockade of enough viral mRNAs produced by the early viral RNA.

The antiviral state induced in HeLa cells after treatment with HuIFN-a (Ly) blocks viral protein synthesis as analyzed by polyacrylamide gels very efficiently. A phenomenology similar to that reported for vaccinia-infected L cells treated with interferon is apparent. A drastic inhibition of cellular protein synthesis develops in vaccinia-infected L cells when they are grown in suspension and pretreated with interferon with no detection of viral protein synthesis [12,13]. However, in that system, the synthesis of viral RNA is stimulated a lot

[13]. This result contrasts with that obtained for poliovirus in which viral RNA synthesis is inhibited.

According to present models on the molecular mechanism of interferon action, two different modes of inhibition of viral replication are proposed [26]. One is the stimulation of a nuclease activity mediated by the synthesis of several adenosine oligonucleotides : the (2’-5’)A, system. The other is the phosphorylation of the initiation factor eIF-2 involved in the binding of the initiator Met-tRNA, to the small ribosomal subunit [27]. Both are triggered by the presence of viral dsRNA in cell-free systems. Unpublished results from our laboratory indicate that cellular mRNAs are in a translatable form in cells treated with interferon and poliovirus, four hours post infection. If this is so, the (2’-5’)A, system could play no part in the inhibition of cellular and viral protein synthesis observed. Similar results have already been reported for mengovirus-infected cells treated with mouse interferon [28]. It still remains to be established whether the phosphorylation of eIF-2 is the mechanism that blocks replication of poliovirus in interferon-treated HeLa cells. Studies to measure the degree of phosphorylation and the activity of this initiation factor are in progress in our laboratory. Infection by vesicular stomatitis virus or Mengo virus does not increase the level of phos- phorylation in interferon-treated L cells. This only occurs when cells are incubated with the synthetic dsRNA poly(r1). poly(rC) [29]. Recently, the same authors have reported opposite results with reovirus [30].

M. A. Ramos is thanked for expert technical assistance. Comisidn Asesora de Investigacidn Cientqica y TPcnica, Fundacidn Cient fica de la Asociacidn Espaiiola contra el Cancer and Fondo de lnvestigaciones Sanitarias de la Seguridad Social provided financial support.

629

REFERENCES

1. Stewart, W. W. 11. (1981) The Interferon System, 2nd edn,

2. Lengyel, P. (1982) Annu. Rev. Biochem. 51, 251 -282. 3. Sen, G. C. (1982) Progress in Nucleic Acid Research and Molecular

4. Levy, H. B. (1964) Virology, 22, 575 -579. 5. Gauntt, C. J. & Lockart, R. Z., Jr (1968) J . Virol. 2, 567-578. 6. Kerr, I. H., Friedman, R. M., Esteban, M., Brown, R. E., Ball, L.

A,, Metz, D. H., Rigby, D., Tovell, D. R. & Sonnabend, J. A. (1973) Adv. Biosci. 11, 109-126.

Springer-Verlag, Wien und New York.

Biology, 27, 105 -156.

7. Falcoff, R. & Sanceau, J. (1979) Virology, 98, 433-438. 8. Muhoz, A. & Carrasco, L. (1981a) J . Gen. Virol. 56, 153-162. 9. Agol, V. I. (1980) Prog. Med. Virol. 26, 119-157.

10. Kitamura, N., Semler, B. L., Rothberg, P. G., Larsen, G. R., Adler, C. J., Dorner, A. J., Emini, E. A,, Hanecak, R., Lee, J. J., Van Der., Were, S., Anderson, C. W. & Winner, E. (1981) Nature, 291, 547 -553.

11. Muboz, A. & Carrasco, L. (1981b) Intervirology, 16, 106-113. 12. Joklik, W. K. & Merigan, T. C. (1966) Biochemistry, 56,558 -565. 13. Metz, D. H. & Esteban, M. (1972) Nature, 238, 385 -388. 14. Carrasco, L. &Smith, A. E. (1980) Pharmacol. Ther. 9,311 - 3 5 5 .

15. 16. 17. 18.

19.

20. 21. 22. 23.

24. 25.

26. 27.

28.

29. 30.

Kohn, A. (1979) Adv. Virus Res. 24, 223 -276. Pasternak, C. A. & Micklem, K. J. (1981) Biosci. Rep. 1,431 -448. Carrasco, L. & Smith, A. E. (1976) Nature, 264, 807-809. Egberts, E., Hackett, P. B. & Traub, P. (1977) J . Virol. 22,

Nair, C. N., Stowers, J. W. & Singfield, B. (1978) J . Virol. 31,

Lacal, J. C. & Carrasco, L. (1982) Eur. J. Biochem. 127,359-366. Carrasco, L. (1978) Nature, 272, 694-699. Nair, C. N. (1981) J . Virol. 37, 268-273. Lacal, J. C. & Carrasco, L. (1983) J . Gen. Virol 64, 787-

Contreras, A. & Carrasco, L. (1979) J . Virol. 2Y, 114-122. Contreras, A., Vazquez, D. & Carrasco, L. (1978) J . Antibiot.

Baglioni, C. (1979) Cell, 17, 255-264. Williams, B. R. G. & Kerr, I. A. (1980) Trends Biochem. Sci. 5 ,

Vaquero, C., Aujeau-Rigaud, O., Sanceau, J. & Falcoff, R. (1981)

Gupta, S. L. (1979) J . Virol. 19, 301 -311. Gupta, S. L., Holmes, S. L. & Mehra, L. L. (1982) Virology, 120,

591 -597.

184 - 189.

794.

(Tokyo) , 31, 598 -602.

138-140.

Antiviral Res. I , 123 - 134.

495 -499.

A. Muiioz and L. Carrasco, Departamento de Microbilogia, Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas y Universidad Aut6noma de Madrid, Canto Blanco, Madrid-34, Spain