Vol. 65, No. 8

JOURNAL OF VIROLOGY, Aug. 1991, p. 4130-4136

0022-538X/91/084130-07$02.00/0 Copyright © 1991, American Society for Microbiology

Posttranscriptional Regulation of Beta Interferon Expression in Erythroid Friend Cells Treated with Gamma Interferon GIOVANNA MARZIALI,1 GIANNA FIORUCCI,1'2 ELIANA M. COCCIA,1 ZULEMA PERCARIO,1 JACOB RABER,3 ANGELA BATTISTINI,l GIOVANNI B. ROSSI,' ELISABETTA AFFABRIS,J4 AND GIOVANNA ROMEO12* Laboratorio di Virologia, Istituto Superiore di Sanitac,' and Istituto di Tecnologie Biomediche CNR,2 Rome, and Istituto di Microbiologia, Universita di Messina, Messina, Italy, and Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel3 Received 4 February 1991/Accepted 1 May 1991

Treatment of Friend erythroleukemia cells (FLC) with

gamma

interferon (IFN-y) in the

presence

of

anti-IFN-13 antibodies reduces the effectiveness of the antiviral state and the induction of 2'-5'-oligoadenylate

synthetase activity, indicating that the antiviral activity of IFN-,y in FLC is in part mediated by the production of IFN-i. Accordingly, IFN-y induces a less pronounced antiviral state in FLC resistant to IFN-a$/, than in wild-type cells. Moreover, while results of run-on assays indicate that both IFN-ot and -,1 genes are constitutively transcribed in these cells, FLC treatment with IFN--y induces only IFN-I mRNA accumulation. These results indicate that posttranscriptional mechanisms are involved in the regulation of IFN-,I and -a expression by IFN-y. The low amounts of the induced IFN-I synergize with IFN-y in mounting the potent antiviral effect. MATERIALS AND METHODS

Three types of interferon (IFN), IFN-ot, -P, and --y, are recognized, depending on the nature of the producing cells and the stimulus that induces their production. IFN-a and -I8 (type I IFN) are chemically and genetically very similar and appear to share at least one common cell surface receptor in both human and mouse cells (7, 32). IFN--y (type II IFN) is quite distinct from type I IFN since is produced by large granular and T lymphocytes stimulated with mitogens or specific antigens and appears to be involved in immune and inflammatory responses (56, 60). Both type I and type II IFN induce a state of resistance to viral replication and a reduced cell proliferation in susceptible cells. They are unrelated by primary sequence and act through distinct cell surface receptors (3, 5, 7, 51, 53) to induce expression of partially overlapping sets of genes (58). However, by acting in concert they can produce synergistic interactions leading to mutual reinforcement of the physiological response (14, 20-22, 37, 41, 61). The mechanisms by which type I and type II IFN induce gene expression are somewhat different, since protein synthesis inhibitors block induction of some genes by IFN--y while not affecting the response to IFN-a (17, 35, 36, 40). It is therefore possible that in the first round of protein synthesis, IFN--y induces the production of a substance which in turn induces the antiviral state. In this respect, it has been reported that IFN--y is able to induce the production of type I IFN in some cell types, such as L929 murine cells (30, 31) and macrophages (24). Here we report that IFN--y induces the production of IFN-P in Friend erythroleukemia cells (FLC). This effect is mediated by a posttranscriptional regulatory mechanism. In addition, the induced production of IFN-,B is not totally responsible of the antiviral state induced by IFN--y, since IFN-ot/p-resistant cells still exhibit a reduced but detectable antiviral state after IFN--y treat-

Cell cultures and IFNs. FLC and L929 cells were grown in RPMI 1640 supplemented with 5% fetal calf serum, penicillin (100 IU/ml), and streptomycin (100 ,ug/ml). Cell mortality was evaluated by the trypan blue dye exclusion method and never exceeded 5%. Murine IFN-a/, (MuIFN-o/,B) prepared and purified as described previously (11) to 107 U/mg of protein was used. Recombinant IFN--y (107 U/mg of protein) was produced by Genentech and kindly provided by Boehringer Ingelheim. Amounts of MuIFNs are given in laboratory units, i.e., the amount of IFN reducing the plaque titer of vesicular stomatitis virus (VSV) by 50%. This unit equals 4 reference units of the National Institutes of Health standard G-002-904-511. Antibodies. Sheep anti-MuIFN-ao3/ globulin (sheep R5/4; 49) had a neutralizing titer of 10-5 against 4 to 8 U of MuIFN-a/o. The origins of hybridomas producing rat antiMuIFN-o (clone 4E-A1) and anti-MuIFN-P (clone 7F-D3) monoclonal antibodies (MAbs) have been described elsewhere (33). Cells were passaged in ascitic form in BALB/c nude mice. Ascitic fluids were concentrated by ammonium sulfate precipitation. The MAb to MuIFN-ot (clone 4E-A1) had a titer of 2 x 10-3 against 4 U of MuIFN-a. The MAb to MuIFN-P had a titer of 2 x 10-5 to 5 x 10-5 against 4 U of MuIFN-,. Titration of antibodies to IFN was performed as follows. Serial dilutions of antibodies were performed in RPMI medium with 2% fetal calf serum in 96-well culture microplates (Falcon); 50 ,ul of each antibody dilution was added to 50 ,ul of IFN (4 to 8 experimental units) in a different 96-well culture microplate. To titrate MAbs to MuIFN-ot, either purified MuIFN-a from L cells (33) or MuIFN-otl from CHO cells (kindly provided by J. Trapman and E. Zwarthoff) was used; to titrate MAbs to MuIFN-1, electrophoretically pure MuIFN-, from Newcastle disease virus-induced C-243 cells (purified by affinity chromatography using a Sepharose column with MAbs to MuIFN-, [kindly provided by J. Trapman]) was used. IFN and antibodies were incubated at

ment.

*

Corresponding author. 4130

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POSTTRANSCRIPTIONAL ACTIVATION OF IFN-P BY IFN-y

37°C for 90 min before the addition of L cells (20,000 cells per well in 100 RI of RPMI with 2% fetal calf serum). After 24 h at 37°C, L-cell monolayers were infected with VSV (0.1 PFU per cell), and cytopathic effect was observed 24 h later. Polyclonal antibodies against the synthetic peptide B synthesized starting from the cDNA sequence of IFNinduced 2'-5'-oligoadenylate (2-SA) synthetase were kindly provided by Judith Chebath, Weizmann Institute of Science, Rehovot, Israel, and were used to immunoprecipitate the multiple enzyme forms (40, 46, 67, and 100 kDa in human cells and 110, 70, and 43 kDa in mouse cells) (9). Virus. Stocks of VSV (Indiana strain) and of encephalomyocarditis virus (EMCV) were obtained by infecting L929 cell monolayers with a low multiplicity of infection (0.1 PFU per cell) and titrated by plaque assay on the same cells. Titers ranged between 108 and 108-5 PFU/ml. Hybridization assay. Total cellular RNA was extracted as described previously (27). To synthesize complementary RNA probes, plasmid pGEMm-beta-3, containing the 648-bp BamHI-PstI fragment of an IFN-1 cDNA clone (16), was linearized with EcoRI; plasmid pSP64, containing a 610-bp BamHI-HindIII fragment of a murine IFN-a2 cDNA clone, was linearized with BamHI (54). The plasmids were transcribed in presence of [a-32P]UTP (3,000 Ci/mmol) as described previously (26). The RNA was purified by phenol extraction and electrophoresed on 1% low-melting-point agarose gels. The corresponding gel slice was excised and melted at 65°C. The radiolabeled RNA (_106 cpm) was added to 70 to 100 ,ug of cellular RNA dissolved in a solution containing 20 p,l of 80% formamide-0.4 M NaCl-3 mM sodium acetate-1 mM EDTA-20 mM 3-morpholinepropanesulfonic acid buffer (pH 7.0). These reaction mixtures were heated for 5 min at 85°C and then for 18 h at 65°C; 0.2 ml of 30 mM sodium acetate-0.1 M NaCl-2 mM zinc sulfate-5% glycerol-1,500 U of S1 nuclease was added for 45 min at 37°C. The samples were extracted with chloroform and fractionated on 5% polyacrylamide gels with Tris-borate buffer (43). Nuclear run-on transcription assay. Cells collected and washed with ice-cold phosphate-buffered saline were centrifuged at 1,600 x g for 5 min. The cell pellet was suspended in Nonidet P-40 lysis buffer (10 mM Tris [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40), incubated for 5 min on ice, and sheared gently through a 200-,ul Gilson tip. Nuclei were isolated on a 0.7 M sucrose cushion. RNA was labeled with [t_-32P]UTP and purified as described previously (55). Labeled RNA was hybridized to specific cDNA probes immobilized on nitrocellulose filters. An H-2Dd probe was used as control for genes induced by IFN-,y, glyceraldehyde3-phosphate dehydrogenase and P-actin cDNA probes were used for normalization, and pGEM-3 (Promega) served as a negative control for nonspecific hybridization. Filters were incubated in a solution containing 50% formamide, 1% sodium dodecyl sulfate (SDS), 5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5x Denhardt's solution, and 100 ,ug of yeast tRNA per ml at 42°C for 4 days, washed twice in 2x SSC-0.2% SDS for 15 min at room temperature, and then washed twice in 0.2x SSC-0.2% SDS for 30 min at 65°C. Preparation of cell extracts. The postmitochondrial supernatant fraction was prepared at 0 to 4°C from packed cells lysed into 1.5 volumes of homogenization buffer (10 mM Tris-HCl [pH 7.5], 7 mM P-mercaptoethanol, 10 mM KCl, 1.5 mM magnesium acetate, 0.5% Nonidet P-40). The homogenate was centrifuged at 10,000 x g for 10 min. The supernatant (S-10) was either assayed immediately or stored

4131

.-20 U/mi-i

10 U/ Mt 3(n

21-

ZLA 0L

0tn

-1-

ctr

20h 43h -IFN- y i

20h

43h

20h

IFN-af-

FIG. 1. Reduction of virus yield in FLC treated with IFN-a/, or -y. Cells were seeded at 2 x 105/ml, treated with IFNs, and then infected with 4 PFU per cell. Supematants of 2 x 106 cells were collected 8 h later, and virus yields were evaluated by plaque assays on confluent L929 cell

monolayers.

in aliquots at -80°C. Protein concentration was determined by the Bio-Rad protein assay. Western immunoblot analysis. Proteins (40 ,ug) from S-10 cell extracts were heated and electrophoresed in 8.5% SDSpolyacrylamide gels. Electrophoretic transfer onto nitrocellulose paper and reactions with anti-peptide B antibodies and 125I-labeled protein A were performed as described previ-

ously (9). Assay of 2-5A synthetase activity. Proteins (100 ,ug) from S-10 cell extracts were incubated for 1.5 h at 30°C in 20 ,ul of a solution containing 20 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid (HEPES; pH 7.4), 15 mM KCl, 25 mM magnesium acetate, 1 mM dithiothreitol, 5 mM ATP, 4 mM fructose-1,6-diphosphate, 20 ,ug of poly(rI) poly(rC) per ml, and 0.8 ,uCi of [2,8-3H]ATP. The synthesized radioactive 2-SA oligomers were isolated by chromatography on DEAE-cellulose as described previously (46) and counted by liquid scintillation. RESULTS

IFN-y induces the production of IFN-j8 in wild-type (wt) FLC. The antiviral state induced by type I and type II IFN in FLC was evaluated. Cells treated with 10 U of IFN-oa/p or IFN-y per ml for 20 and 43 h were infected with 4 PFU of VSV per cell. Supernatants were collected 8 h later, and virus yields were evaluated by plaque assay. As shown in Fig. 1, cells treated with IFN-y need more than 20 h to exhibit a maximal antiviral state, suggesting that some other mediator(s) might contribute to its establishment. Therefore, the ability of IFN--y to induce type I IFN in FLC was investigated. FLC were treated with IFN--y in the presence of either polyclonal antibodies to MuIFN-a/f or MAbs that neutralize MuIFN-a or -P. Figure 2 shows that treatment of cells with 50 U of IFN--y per ml in the presence of polyclonal antibodies to MuIFN-a/, or of an MAb to MuIFN-, reduces the effectiveness of the antiviral state without abolishing it completely, whereas treatment with IFN--y in the presence of an MAb to IFN-ot does not. This finding suggests that IFN-y induces the production of IFN-,B but not IFN-ot in FLC. Table 1 shows the titration of type I IFN in supernatants of FLC treated for 43 h with IFN--y. Supernatants were treated at pH 2 for 48 h, and then the presence of type I IFN was evaluated by reduction of the EMCV cytopathic effect on L929 cells. Production of type I

4132

MARZIALI ET AL.

J. VIROL.

w.t.

-

(X

P-R

-

6h

3h

3h

6h

2-

(Q)

-IFNct

LuJ z

FIG. 3. Kinetics of appearance of type I IFN mRNAs in FLC treated with IFN--y. RNA extracted from FLC seeded at 106/ml and treated or untreated (-) with IFN--y (500 U/ml) was hybridized with a riboprobe for MuIFN-a or MuIFN-P in a hybridization-in-solution assay. See Materials and Methods.

4-

0a

Li.

0.

-

1

-2Antibodies - + IFNs oc tr

(Xa

+

-

+ a.aa y

ctr

apP

- + y

43 hrs - 50 U/rmn

FIG. 2. Reduction of virus yield in wt and IFN-a/13-resistant

(otp-R) FLC after treatment with IFN-a/f or -,y in the presence of antibodies to type I IFN. Cells were treated as for Fig. 1. Anti-IFNa/ serum (+) or anti-IFN-a (a.a) or -P (a.p) MAbs were added at

cell seeding with IFN--y or preincubated for 30 min at 37°C and 90 min at 4°C with IFN-a/13. Vertical numbers indicate log difference of virus yield between untreated and treated cells.

IFN was very low, but detectable, starting from a 50-U/ml dose of IFN--y. The appearance of type I IFN mRNAs in FLC treated with IFN-y was subsequently evaluated. RNA extracted from FLC treated with 500 U of IFN--y per ml was hybridized with cRNA probes that specifically recognize IFN-a or -I mRNA. In agreement with biological data, Fig. 3 shows that IFN-y treatment induces the appearance of IFN-P mRNA. A 3-h treatment with IFN--y is sufficient to detect the IFN-1 mRNA, and its steady-state level increases after 6 h of treatment. In contrast, IFN-a mRNA is not observed (Fig. 3) even if treatment with IFN--y is prolonged to 12 h (data not shown). IFN-,y induction of an antiviral state in wt and IFN-a/,Bresistant FLC is not mediated only by IFN-13 production. As shown in Fig. 2, VSV yield is still reduced by 2.5 logs in wt cells treated with 50 U of IFN--y per ml in the presence of polyclonal antibodies to MuIFN-cx/1 or of MAbs to MuIFN-1. Accordingly, IFN--y induces a less pronounced antiviral state in FLC (clone 3Cl8) resistant to IFN-a/p (1, 2, 11, 52) than in wt cells (2.8 versus 4.0 logs of reduction in VSV yield) (Fig. 2). To further evaluate this effect, the kinetics of induction of 2-SA synthetase was followed in wt and IFN-ao/g-resistant cells after treatment with type II IFN. 2-5A synthetase is one of the best known markers of IFN

action. Different isoforms have been identified in both human (100, 67, 46, and 40 kDa) and mouse (110, 70, and 43 kDa) cells (9), and their involvement in antiviral activity has been directly demonstrated by transfection and constitutive expression of cloned full-length cDNAs (10, 12). Figure 4A shows that type II IFN treatment induces 2-5A synthetase activity in wt cells at a lower extent than does type I IFN. The simultaneous addition of polyclonal antibodies to type I IFN decreases the IFN-y induction at a level comparable to that observed in IFN-ct/P-resistant FLC treated with IFN-y (Fig. 4B). Accordingly, the induction of the three murine isoforms detected in cell extracts by Western blots was lowered by antibodies to type I IFN in wt cells treated with IFN--y (Fig. 4C). IFN-y acts synergistically with IFN-1 in establishing a powerful antiviral state. Figure 5 shows that treatment of cells with as low as 5 U of IFN--y per ml in the presence of polyclonal antibodies to IFN-o/13 reduces the IFN-,y-induced antiviral state (1.15 versus 2.6 logs of reduction in VSV A

a

R

.B S..

.

I-4 --i

y .j .,

-aC:

C

G O 12 24

;-; 1e o4

929 I FNr-

+

-

6

12

FLC 745 24 46

72

46

24 48kh +

anti IFN nO

10 Kda-

TABLE 1. Titration of type I IFN present in supernatants of cells treated for 43 h with IFN--ya IFN--y treatment (U/ml)

0 5 10 50 400

IFN recovered in cell

(U/mi)

70 Kda-

supernatants 43 Kda-

Total

pH 2 resistant

37 150

9 37

a IFN was evaluated by reduction of EMCV cytopathic effect on L929 cells; a control preparation of IFN--y (200 U/ml) was completely inactivated by treatment at pH 2 for 48 h. -, undetectable.

_

FIG. 4. Kinetics of 2-SA synthetase induction. Shown are enzymatic activities (A and B) and Western blot analysis (C) in FLC treated with IFNs with or without polyclonal antibodies to IFN-a/,. Cell were seeded at 4 x 105/ml. Enzymatic assay and Western blot were performed on cell extracts as described in Materials and Methods.

VOL. 65, 1991

POSTTRANSCRIPTIONAL ACTIVATION OF IFN-j BY IFN--y i- w. t.

4133

-

3LO~

240 qo

II I

N1

=

1-

I 0-

I

*

,I -,

I II

c, -

-.

...'I

-1+ Antibodies i- c tr IFNs

-

+

y

43 hrs-5 U/mi

FIG. 5. Reduction of virus yield in wt FLC after treatment with IFN--y (5 U/ml) in the presence of antibodies to type I IFN. Cells were treated as for Fig. 1. Anti-IFN-a/p serum (+) was added at cell seeding with IFN--y or preincubated for 30 min at 37°C and 90 min at 4°C with IFN-a/P.

yield). The data in Table 1 indicate that 5 U of IFN--y per ml induces undetectable levels of type I IFN, suggesting that the potent antiviral effect (2.6 logs of reduction in VSV yield) observed after 43 h of treatment with IFN--y (Fig. 5) is due to the synergistic interaction between type I and type II IFN. This hypothesis was verified by treating FLC with subliminal doses of both IFN types. Figure 6 shows that treatment of FLC with 1 U of IFN--y or 5 U of IFN-a/,B per ml for 20 or 43 h induces less than a 1-log reduction in VSV yield. Conversely, the simultaneous treatment with both or pretreatment with one type of IFN followed by the other results in >2.5 logs of reduction in VSV yield. Transcription of type I IFN genes in FLC treated with IFN-y. Run-on experiments were performed to investigate the molecular mechanism underlying the induction of IFN-P by IFN--y in FLC. To examine the effect of IFN--y treatment on the transcription of type I IFN genes, the RNA synthesized by nuclei of untreated and IFN--y-treated FLC was hybridized to nitrocellulose-bound linearized plasmid DNAs containing specific sequences to detect IFN-o and IFN-P transcripts, respectively. Surprisingly, transcription of both IFN-a and IFN-P genes was present constitutively in untreated FLC (Fig. 7) and was repeatedly observed; 3-, 6 (Fig. 7)-, and 12 (data not shown)-h treatments with IFN-y do not IFN-aP=5 U/ml; IFN-y=l U/mi

32-

1

~o

0,

cN.

Ho

cL1-1

N

'>

'iio n

0-

-1

y

-

a

-

ap

-

y y

ap

aCL

y

y

-

CL3+y

FIG. 6. Evidence that IFN-y and IFN-a/, act synergistically to produce the antiviral state in FLC. Cells were treated as for Fig. 1. IFNs were added at cell seeding (abscissa, upper line) and/or 20 h later (abscissa, lower line). VSV infection was performed 43 h after cell seeding.

FIG. 7. Transcription assay of IFN-a, IFN-,, and H-2Dd genes in FLC treated with IFN--y. Cells were seeded at 106/ml in the presence of IFN-y. Run-on assays were performed as described in Materials and Methods. GADPH, Glyceraldehyde-3-phosphate dehydrogenase.

further increase transcription of type I IFN genes. On the contrary, IFN--y treatment increases the transcription rate of the H-2 class I (H-2Dd) (Fig. 7) and 2-5A synthetase (data not shown) genes, which are known to be induced transcriptionally by IFN--y. DISCUSSION There is increasing evidence that type II IFN specifically induces the production of type I IFN in certain cell systems. In fact, Hughes and Baron (30, 31) reported that a large component of the antiviral activity of MuIFN--y in L929 cells was due to the production of IFN-a. In addition, Gessani et al. (24) reported that MuIFN--y or lipopolysaccharide promoted accumulation of IFN-I mRNA in murine peritoneal macrophages and the secretion of the corresponding molecule. In contrast, IFN-y does not induce transcription and production of the IFN-1 gene in HeLa cells (48). The results presented in this report demonstrate that also in FLC, MuIFN--y treatment induces the expression of IFN-P mRNA and the production of the corresponding protein. The produced IFN-, synergizes with IFN--y, resulting in the establishment of a powerful antiviral state, in agreement with observations obtained for other cell systems (14, 21, 22, 37, 41). However, the antiviral effect induced by IFN-y treatment of FLC is not completely mediated by the induced production of IFN-P. In fact, IFN--y is able per se to elicit a reduced but significant protection to virus infection in FLC resistant to type I IFN (clone 3Cl8) (1, 2, 11, 52) in comparison with wt FLC. These data are in agreement with the observation of de novo transcription of 2-5A synthetase genes from nuclei of type I IFN-resistant FLC treated with IFN--y and the corresponding formation of a specific interferon-responsive sequence-protein complex (Fg) in cell extracts (13). Recently, Lewis et al. (41) suggested that IFN--y induces the synthesis of a protein which can act synergistically with a signal produced by the IFN-P receptor. Levy et al. (39) observed that IFN-y induces the synthesis of one of the two subunits of the positive transcription factor ISGF3, ISGF3-y, subsequently activated in response to IFN-a treatment. The cooperative induction of cytokine-specific transcription factors is one of the mechanisms hypothesized for

J. VIROL.

MARZIALI ET AL.

4134

reinforcing effects of distinct cell surface ligands while still maintaining the specificities of the individual inducers. The presence of a protein-binding site (PRD-1) in the promoter region of the IFN-P genes (homologous to the central core of the interferon-responsive sequence present in the promoter region of 2-5A synthetase and other IFNinduced genes) (18, 23, 28, 34, 48, 57) has been postulated. This raises the possibility that IFN--y initially induces the simultaneous expression of the 2-5A synthetase genes and of the IFN-,3 gene via a similar transduction pathway. Subsequently, the signal transduction pathway induced by the IFN-, gene reinforces the expression of genes responsive to both type I and type II IFN. For this reason we investigated the induction of type I IFN, H-2Dd, and 2-5A synthetase gene expression by run-on assays. As expected, IFN--y increases the transcription rate of the H-2 class I histocompatibility H-2Dd and 2-5A synthetase genes but, surprisingly, does not modify the transcription of the IFN-aL genes, the expression of which is constitutive in and untreated cells. These results suggest that IFN-y activates in FLC some unknown posttranscriptional regulatory mechanisms that lead to the stabilization of IFN-P but not IFN-a mRNA. The IFN-, gene is turned on and off mainly at the level of transcription through the interaction of protein factors with positive and negative regulatory sequences of its promoter (29, 38, 44, 45). On the other hand, the presence of mechanisms of posttranscriptional regulation of the human IFN-,B gene has been already suggested (47, 50). In addition, determinants for mRNA degradation in both the 3' untranslated region and the region 5' to the translation stop codon of human IFN-P mRNA have been identified (59). Gessani et al. (25) observed that transcription of the IFN-13 gene is elevated in murine peritoneal macrophages without accumulation of IFN-,B mRNA. IFN-y-induced production of IFN-P in these cells is probably a consequence of an unidentified posttranscriptional regulatory mechanism. This finding supports our data for FLC. The induction of different genes in response to IFN--y is variable. The speed of the response varies from extremely rapid (19) to very slow (4, 6). The response can be transient (19, 42) or sustained for several days (4, 6, 8, 15). Protein synthesis may be required (4, 6, 8), partially required (40), or unnecessary (15, 19) for induction. Therefore, it appears that there are several ways in which IFN-y can cause changes in gene expression in any given cell type. With respect to IFN-P induction, IFN--y appears unable to induce IFN-f production in HeLa cells, in which constitutive transcription of the IFN-P gene is undetectable before and after IFN-y treatment (48). It may be speculated that IFN--y is able to induce type I IFN production only by acting at a posttranscriptional level in cells in which transcription of the gene is already present. -I

REFERENCES 1. Affabris, E., C. Jemma, and G. B. Rossi. 1982. Isolation of interferon-resistant variants of Friend erythroleukemia cells: effects of interferon and ouabain. Virology 120:441-452. 2. Affabris, E., G. Romeo, F. Belardelli, C. Jemma, N. Mechti, I. Gresser, and G. B. Rossi. 1983. 2-5A synthetase activity does not increase in interferon-resistant Friend leukemia cell variants treated with alpha/beta interferon despite the presence of highaffinity interferon receptor sites. Virology 125:508-512. 3. Aguet, M., F. Belardelli, B. Blanchard, F. Marcucci, and I. Gresser. 1982. High affinity binding of 125I-labeled mouse interferon to a specific cell-surface receptor. IV. Mouse gamma interferon and cholera toxin do not compete for the common receptor site of alpha/beta interferon. Virology 117:541-544. 4. Amaldi, I., W. Reith, C. Berte, and B. Mach. 1989. Induction of HLA class II genes by IFN-gamma is transcriptional and requires a trans-acting protein. J. Immunol. 142:999-1004. 5. Anderson, P., Y. K. Yip, and J. Vilcek. 1982. Specific binding of 1251-human interferon-gamma to high affinity receptors on human fibroblasts. J. Biol. Chem. 257:11301-11304. 6. Blanar, M. A., E. C. Boettger, and R. A. Flavell. 1988. Transcriptional activation of HLA-DR-alpha by interferon gamma requires a trans-acting protein. Proc. Natl. Acad. Sci. USA 85:4672-4676. 7. Branca, A. A., and C. Baglioni. 1981. Evidence that types I and II interferons have different receptors. Nature (London) 294: 768-770. 8. Caplan, H. S., and S. L. Gupta. 1988. Differential regulation of a cellular gene by human interferon-alpha and interferongamma. J. Biol. Chem. 263:332-339. 9. Chebath, J., P. Benech, A. Hovanessian, J. Galabru, and M. Revel. 1987. Four different forms of interferon-induced 2',5'oligo(A) synthetase identified by immunoblotting in human cells. J. Biol. Chem. 262:3852-3857. 10. Chebath, J., P. Benech, M. Revel, and M. Vigneron. 1987. Constitutive expression of (2'-5')oligo A synthetase confers resistance to picornavirus infection. Nature (London) 330:587-

588. 11. Coccia, E. M., M. Federico, G. Romeo, E. Affabris, F. Cofano, and G. B. Rossi. 1988. Interferons-alpha/beta resistant Friend cell variants exhibiting receptor sites for interferons but no induction of 2-5A synthetase and 67K protein kinase. J. Interferon Res. 8:113-127. 12. Coccia, E. M., G. Romeo, S. Nissim, G. Marziali, R. Albertini, E. Affabris, A. Battistini, G. Fiorucci, R. Orsatti, G. B. Rossi, and J. Chebath. 1990. A full-length murine 2-SA synthetase cDNA transfected in NIH-3T3 cells impairs EMCV but not VSV replication. Virology 179:228-233. 13. Coccia, E. M., D. Vaiman, J. Raber, G. Marziali, G. Fiorucci, R. Orsatti, B. Cohen, N. Nissim, G. Romeo, E. Affabris, J. Chebat, and A. Battistini. 1991. Protein binding to the interferon response enhancer correlates with interferon-induction of 2-5A synthetase in normal and interferon-resistant Friend cells. J. Virol. 65:2081-2087. 14. Czarniecki, C. W., C. W. Fennie, D. B. Powers, and D. A. Estell. 1984. Synergistic antiviral and antiproliferative activities of

15.

16. ACKNOWLEDGMENTS

We thank Judith Chebath for helpful discussions, Sandra Gessani for generous gifts of pGEM-IFN-, and pSP64-IFN-a2, Ramona Ilari for technical assistance, and Sabrina Tocchio for editorial assis-

17.

tance.

This work was supported by grants from the Italy-USA Program Therapy of Tumors, Associazione Italiana Ricerca sul Cancro, Ministero Pubblica Istruzione, and the AIDS project of the Ministry of Public Health, Rome.

18.

on

19.

Escherichia coli-derived human alpha, beta, and gamma interferons. J. Virol. 49:490-496. Decker, T., D. J. Lew, Y. S. E. Cheng, D. E. Levy, and J. E. Darnell, Jr. 1989. Interactions of alpha- and gamma-interferon in the transcriptional regulation of the gene encoding a guanylate-binding protein. EMBO J. 8:2009-2014. Di Maio, D., R. Treishad, and T. Maniatis. 1982. Bovine papillomavirus vector that propagates as a plasmid in both mouse and bacterial cells. Proc. Natl. Acad. Sci. USA 79:40304034. Faltynek, C. R., S. McCandless, J. Chebath, and C. Baglioni. 1985. Different mechanisms for activation of gene transcription by interferons alpha and gamma. Virology 144:173-180. Fan, C. E., and T. Maniatis. 1989. Two different virus-inducible elements are required for human beta-interferon gene regulation. EMBO J. 8:101-110. Fan, X., G. R. Stark, and B. R. Bloom. 1989. Molecular cloning

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23.

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