FEMS Microbiology Letters 21 (1984) 105-111 105 Published by Elsevier

Comparison of the antiviral action of different human interferons against DNA and RNA viruses

(Interferon; herpes virus; poliovirus; antiviral activity)

Alber to Mufioz and Luis Car rasco

Departamento de Microbiologia, Centro de Biologia Molecular, Facultad de Ciencias, Unioersidad A utdnoma, Canto Blanco, Madrid-34, Spain

Received 2 August 1983 Accepted 27 September 1983

1. SUMMARY

Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon a from human buffy coats, interferon fl from human fibroblasts, interferon y from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-a (Ly) and cloned a 2 interferon produced by Escherichia coli containing the human gene for interferon a 2. All preparations were able to pro- tect monolayers of HeLa cells against HSV-1 in- fection when low multiplicities were used. The five IFN preparations were also tested against en- cephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV).

2. INTRODUCTION

Interferons are a family of proteins which are synthesized by cells after stimulation with a given inducing agent. They exert a number of biological activities, including an antiviral effect which has been widely documented [1]. Addition of inter-

feron to cultured cells induces an antiviral state that confers transient resistance to subsequent in- fection by a number of animal viruses [1].

Different interferon molecules are now known. Three major antigenic types of human interferon (HulFN) have been described: HulFN-a, HulFN- fl and HulFN-'y. Interferons of the a type are synthesized by human leukocytes in response to viruses or dsRNA. At least eight to twelve differ- ent HuIFN-a syntheses exist [2]. HuIFN-fl is synthesized by fibroblasts after treatment with dsRNA and there may also be multiple subtypes of this IFN [3,4]. HuIFN-y is synthesized by lymphocytes when they are incubated with anti- gens or mitogens. Comparative studies of all these interferons to determine their relative potency as antiviral agents, as antiproliferative agents and as immunomodulators are now in progress in a num- ber of laboratories. Thus, HuIFN-y was initially reported to possess a more potent antiproliferative activity than HuIFN a and fl [5]. However, later results indicated that it had biological effects equivalent to those of the other types [6-8]. In the present study we have analysed the action of five different human interferons against both DNA and RNA animal viruses.

0378-1097/84/$03.00 © 1984 Federation of European Microbiological Societies

106

3. MATERIALS AND METHODS

3.1. Cells and viruses

HeLa cells, L929 cells, and BHK-21 cells were grown in Dulbecco's modified Eagle's (E4D) medium supplemented with 10% newborn calf serum (Gibco) (E4D10 medium) and incubated at 37°C in a 5% CO 2 atmosphere.

VSV was grown on BHK-21 cells in E4D medium supplemented with 2% newborn calf serum (E4D2). Poliovirus type I and HSV-1 were grown on HeLa cells in the same medium. EMC virus was grown on L929 cells in a mixture of Eagle's medium, phosphate-buffered saline (PBS) solution and E4D medium (80 /15 /5E) supplemented with 1% newborn calf serum.

For VSV, EMC and poliovirus, the fraction obtained after removal of cell debris by low-speed centrifugation was used as a source of seed virus. In the case of HSV-1, cells were removed 22 h postinfection and the virus released from cells by successive cycles of freezing and thawing. The fraction obtained after removal of cell debris by low-speed centrifugation was used as a source of virus.

3.2. Interferons

Partially purified human leukocyte interferon was obtained from Dr. K. Cantell (Finland) (1.0. 106 I U / m g protein), HulFN-a (Ly) (1.6- 106 IU// mg protein) from Drs. Finter, Fantes and John- ston, Wellcome Research Laboratories, Becken- ham (England); recombinant HulFN-a 2 produced by E. coli (7 .10 7 I U / m g protein) from Dr. C. Weissman (Zurich); HulFN-fl (1.0.106 I U / m g protein) and HulFN-y (1.0-104 I U / m g protein) were given by Dr. P~rez Aranda (Antibiotics Laboratories, Madrid).

3.3. Virus infection and measurement of protein synthesis

HeLa-cells grown on 16-mm diam. 24-well plates (Falcon Plastics) were infected with the particular virus at the m.o.i, described in each experiment. After 1 (EMC, VSV, polio) or 1.5

(HSV-1) h incubation at 37°C the medium was removed and 1 ml E4D2 medium was added. The point of virus addition was considered as - 1 h and that of virus removal as 0. Incubation was continued at 37°C until labelling. For this purpose 0.5 ml methionine-free E4D1 medium and 0.11 luCi [35S]methionine (1100 Ci /mmol; 5.4 mCi /ml ; The Radiochemical Centre, Amersham, U.K.) were added to the cells for 1 h. The medium was then removed, the cells were washed with PBS and precipitated with 5% TCA. After 5 min the TCA was removed and the cell monolayer washed three times with ethanol, dried under an infrared lamp and dissolved with 250 /d 0.1 N NaOH plus 1% SDS. A 125-/~1 sample was counted in an Inter- technique scintillation spectrometer.

3.4. Analysis by polyacrylamide gel electrophoresis (PAGE) of the proteins synthesized in virus-infected cells

HeLa cells were grown on 30-mm petri dishes and labelled with 5.4 #Ci [35S]methionine. At the end of the labelling period the cells were washed with 1 ml PBS solution and dissolved in 200 IH 0.02 N NaOH plus 1% SDS and 200 /~1 sample buffer (62.5 mM Tris pH 6.8; 2% SDS; 0.1 M di th io thre i to l ; 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 /~1 were applied to a 15% poly- acrylamide gel and run overnight at 30 V. Fluorog- raphy of the gel was carried out with, 2,5-diphen- yloxazole-dimethyl sulphoxide (20%, w/w). The dried gels were exposed using XS-5 X-ray films (Kodak).

4. RESULTS

4.1. Effect of different interferons against HS V-1

Five interferon preparations were used: IFN-a obtained from buffy coats (IFN-a (Le)), IFN-fl from human cultured diploid fibroblasts, IFN-7 from human lymphocyte-enriched populations in- duced with PHA, IFN-a (Ly) from lymphob- lastoid Namalva cells and IFN-a 2 produced by E.

107

,ooL' o ' 'l'b ' 'I'c ' 'i'd ' '.I. ' e ' ' Jmo-J

50 * 50

:

4 40 400 4 40 400 4 40 400 4 40 400 4 40 400

IFN (U/ml)

Fig. 1. Protection of HeLa cells by different human interferon against HSV-1 infection. Cells grown on 24-well plates were treated 18 h before infection with the indicated concentration of (a) HuIFN-a (Le); (b) HuIFN-fl; (c) HuIFN-T; (d) HuIFN-a (Ly), or (e) HuIFN-a 2 cloned in E. coll. 48 h after HSV-1 infection the level of protein synthesis was estimated by one hour pulse with [35S]methionine. Uninfected cells treated with each interferon concentration were taken as controls.

coli carrying a plasmid containing the cloned gene of IFN-a z. The source and specific activity of each interferon preparation is given in MATERIALS AND METHODS. The interferons were first titrated against EMC virus as already described [8].

We first tested the protective effect of the five different human interferons on HeLa cells infected with HSV-1. Cells were treated 'overnight with different concentrations of the titrated interferon and then inoculated with HSV-1 at different m.o.i. After 2 days of incubation the cytopathic effect was recorded and the level of protein synthesis estimated. Fig. 1 shows that the most potent inter- ferons against HSV-1 were HulFN-a(Le) and /3. The least effective protection was obtained with cloned HulFN-%. HulFN-y and HulFN-,~(Ly) had intermediate activity. All of the interferons were only able to protect the cell monolayer against HSV-1 infection when low m.o.i.'s were used.

Little is known about the molecular mechanism of the antiherpetic action of interferon. We re- cently found that treatment of HeLa cells with IFN-a (Ly) did not prevent the first round of replication of the virus (unpublished results). Thus herpes virus protein synthesis takes place in the interferon-treated cells at levels almost comparable to those in control cells. To establish wheter a similar effect is obtained with the different inter- ferons listed above, the experiment shown in Fig. 2 was carried out. Cells treated with a high inter- feron dose (400 U /ml ) , were infected with HSV-1 at an m.o.i, of 5 pfu /ce l l and both the levels of protein synthesis and the different proteins synthesized throughout infection were analyzed.

The time course of translation was approximately the same in control and interferon-treated cells with a general but not drastic reduction in protein synthesis in interferon-treated cells particularly at 6 h postinfection.

Analysis of the proteins by PAGE, revealed some quantitative and qualitative differences be- tween control, HSV-1 infected cells, and inter- feron-treated cells. There was a general reduction in viral translation late in infection when the cells had been pretreated with interferon. This reduc- tion was more pronounced when IFN-7 was used. The synthesis of some viral proteins, for example the 18K protein, was inhibited in interferon-treated cells (particularly using IFN-y). These results sug- gest that, although all the interferon preparations allowed the synthesis of the bulk of late viral proteins, the synthesis of some specific viral pro- teins was blocked by all of them, though the different types of interferon had slightly different inhibitory activities.

4.2. Effect of different interferons on the replication of RNA viruses

Interferon is able to inhibit the replication of many unrelated viruses, but each virus differs in its sensitivity in each particular animal cell line [1].

Fig. 3 shows the action of the five different interferon preparations against EMC virus and VSV in HeLa cells. All five were very active against EMC replication.

IFN-a(Le) and/3 were able to protect partially even when high m.o.i.'s were used. The most potent

1 0 8

Time

hr p.i.

Confrol + Hu IFN (400 Ulml)

2 4 6 8 14 2 4 8 8 1 4 2 4 6 8 14 2 4 6 8 "14 7' 4 6 8 1 4 2 4 6 8 14

- - 135

- - 122

- - 6 7

4 2

- - 27.5

- - 1 8

A

b x

E u

0

U

¢- .m ¢- 0

E i

:50

2 0

lO

r I I I I

%

0 + i t + I I t

0 2 4 6 8 14

TIME (ht)

Fig. 2. Effect of treatment with different human interferons on the proteins synthesized in HSV-1 infected HeLa cells. Interferon treatment was carded out 18 h before the infection, the level of protein synthesis was measured and the proteins analyzed at the times indicated as described in MATERIALS AND METHODS. O - - - - - - O, Control untreated cells; • • Hu IFN-a; • • , Hu IFN-fl; I . II, Hu IFN-y; ~ . ~ , Hu IFN-a (Ly); • • , Hu IFN-a 2 cloned in E. coli.

i n t e r f e r o n p r e p a r a t i o n a g a i n s t V S V was I F N -

a ( L y ) , w h e r e a s H u l F N - y w as t he l eas t ac t ive .

W e r e c e n t l y s h o w e d tha t , w h e n H e L a cel ls a re

t r e a t e d w i t h i n t e r f e r o n a n d i n f e c t e d w i t h E M C

v i r u s a t low or h i g h m.o. i . , n o v i ra l p r o t e i n s y n t h e s i s

is o b s e r v e d [9]. T h i s is in a g r e e m e n t w i t h o b s e r v a -

t i o n s f r o m o t h e r l a b o r a t o r i e s [10]. T h e s e r e su l t s

c o n t r a s t w i t h t he a c t i o n of i n t e r f e r o n o n the rep l i -

109

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o --~ '-------#' 4

h i j

40 400 4 40 400 4 40 400 4 40 400 40 400

I00

5O J o 0c

0 ~-

100 ~o

50

IFN (Ulml)

Fig. 3. Antiviral state induced by different human interferons in HeLa cells against EMC virus infection (panels a-e) or VSV infection (panels f-j). Cells grown on 24-well plates were treated with interferon 18 h before infection, and 24 h later the level of protein synthesis was measured in an hour pulse with [ 35 S]methionine. The values are expressed as percentage of protein synthesis referred to that of control uninfected cells treated with the corresponding interferon concentration. (a and f) Hu IFN-a; (b and g) Hu IFN-fl; (c and h) Hu IFN-y; (d and i) Hu IFN-a (Ly); (e and j) Hu IFN-a 2 cloned in E. coli.

cation of HSV-1 described above. As expected, infection of HeLa cells with poliovirus lead to a drastic inhibition of cellular protein' synthesis (Fig. 4). This inhibition was not prevented in cells pre- treated with interferon. Indeed, the inhibition of protein synthesis under those conditions was in some cases even more severe. This effect was par- ticularly marked in cells treated with I F N - y where 2 h after infection, protein synthesis amounted only about 30% of that in untreated poliovirus-in- fected cells. As with E MC virus, no poliovirus protein synthesis was detected in cells pretreated with all five interferons.

5. D I S C U S S I O N

Recent work has shown that a large number of different types and subtypes of interferons are made by human cells. Since interferon is a poten- tial antiviral agent it is interesting to compare the activity of the different interferon molecules.

Our compar ison of the antiherpetic action of five different interferon preparat ions indicates that I F N - a and fl are the most potent as measured by the protection of the cell monolayer against infec- tion by HSV-1. Analysis of the proteins synthe-

sized in IFN-t rea ted cells after infection with HSV-1 shows that almost all late proteins appear in IFN-t rea ted cells though some of them are clearly inhibited. However, no correlation is ob- served between the inhibition of the synthesis of these viral proteins and the extent of cellular pro- tection given by the interferon (Fig. 1). This is in agreement with our previous findings that the main effect of interferon on herpes virus infection oc- curs late in the viral cycle (unpublished results).

The antiviral action of interferon against picor- na virus infection is quite different f rom that described for herpes virus. In the case of polio virus no viral proteins are synthesised throughout infection when the cells were pretreated with inter- feron. All interferons tested prevented the ap- pearance of polio virus proteins but not the cyto- pathic effect and all the cells were eventually killed by virus infection.

In conclusion, our comparat ive studies on the action of different interferon preparat ions against HSV-1 and RNA-conta in ing viruses indicate that al though there are some differences in their potency against some particular virus, all seem to act in the same way: that is all prevented the synthesis of poliovirus proteins and allowed the synthesis of herpes virus proteins.

110

P o l i o v i r u s (10 P F U / c e l I )

Time hr p.i

Control

1 2 4 6

_/NCPVlo _~NCPV]b

NCPV2

- - V P O

- - V P 3

0

b r - -

X

4 0

"ID

0

u ._~ ® 2 0 ¢..

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E |

~ 0

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1 2 4 6 1 2 4 6 1 2 4 6 12 4 6

J i i I

o

\ \

2 4 6 8

T I ME ( h r )

cx2(coli)

1 2 4 6

Fig. 4. Analysis of the polypeptides synthesized in polio virus-infected HeLa cells by polyacrylamide gel electrophoresis following treatment with 200 U / m l of the five different human interferons 18 h before infection. Conditions of infection, measurement and analysis of the proteins synthesized were as described in MATERIALS A N D METHODS. O, Control untreated cells; o, Hu IFN-a; ,t, Hu IFN-fl; II, HU IFN-7; , , Hu I F N - a (Ly); T, Hu IFN-a 2 cloned in E. coli.

A C K N O W L E D G E M E N T S

W e t h a n k Drs. K. Cante l l , N.B. F in te r , K .H. , Fan tes , M.D. J o h n s t o n , C. W e i s s m a n a n d A. P e r e z - A r a n d a for their generous gifts of i n t e r f e ron p repa ra t ions .

The exper t technica l ass is tance of Ms. M.A. R a m o s is acknowledged . We also t h a n k C A I C Y T , ' F u n d a c i b n Cien t i f i ca de la Asoc iac ibn Espaf io la

C o n t r a el Cance r ' a n d ' P l a n C o n c e r t a d o de In- ves t igac ibn No. 5 / 7 8 for their f inanc ia l suppor t .

R E F E R E N C E S

[1] Stewart, W.E., II (1981) The interferon system, 2nd ed., Springer-Verlag, Berlin.

[2] Rubinstein, M., Levy, W.P., Moschera, J.A., Lai, C.Y., Hershberg, R.D., Bartlett, R.T. and Pestka, S. (1981) Arch. Biochem. Biophys. 155,202-212.

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[3] Weissenbach, J., Chernajowsky, Y., Zeevi, H., Schulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. and Revel, M. (1980) Proc. Natl. Acad. Sci. USA 77, 7152-7156.

[41 Seghal, P.B. and Sagar, A.D. (1980) Nature 288, 95-97. [5] Blalock, J.E., Georgiades, G.A., Langford, M.P. and John-

son, H.M. (1979) Cell. Immun. 49, 390-394. [6] Rubin, B.Y. and Gupta, S.L. (1980) Proc. Natl. Acad. Sci.

USA 77, 5928-5932. [7] De Ley, M., Van Damme, J., Claeys, H., Weening, H.,

Heine, J.W., Billiau, A., Vermylen, C. and De Somer, P. (1980) Eur. J. Immunol. 10, 877-883.

[8] Bourgeade, M.F., Chancy, C. and Merigan, T.C. (1980) J. Gen. Virol. 46, 449-454.

[9] Mufioz, A. and Carrasco, L. (1981) J. Gen. Virol. 56, 153-162.

[10] Williams, B.R.G., Golgher, R.R., Hovanessian, A.G. and Kerr, I.M. (1980) in Developments in Antiviral Therapy (Collier, L.H. and Oxford, J., Eds.), pp. 173-187. Academic Press, London.