A full-length murine 2-5A synthetase cDNA transfected in NIH-3T3 cells impairs EMCV but not VSV replication

179,228-233

VIROLOGY

(1990)

A Full-Length

Murine 2-5A Synthetase cDNA Transfected Impairs EMCV but Not VSV Replication

in NIH-3T3

Ceils

ELIANA M. COCCIA,* GIOVANNA ROMEO,*+ AHUVA NISSIM,+ GIOVANNA MARZIALI,* ROBERTO ALBERTINI,*$ ELlSABElTA AFFABRIS, *.I ANGELA BA-iTISTINI,* GIANNA FIORUCCI,**t ROBERTO ORSAlTI,* GIOVANNI B. ROSSI,*,’ AND JUDITH CHEBATH+ *Laboratorio

di Virologia, lstituto Weizmann

Superiore Institute,

di Sanitci, Rome, Italy; tlstituto di Tecnologie Biomediche CNR, Rome, Italy; #Department Rehovot, Israel; glstituto di Patologia Speciale Medica, Universits di Parma, Italy; and 1lstituto di Microbiologia, UniversitG di Messina, Italy Received

January

29, 1990; accepted

of Virology

July 2, 1990

Treatment of cells with interferons (IFNs) induces resistance to virus infection. The 2’-5’oligo A (2-5A) synthetase/ RNase L is one of the pathways leading to translation inhibition induced by IFN treatment. A murine cDNA encoding the 43-kDa 2-5A synthetase was cloned and sequenced. NIH-3T3 cell clones transfected with this cDNA expressed the enzymatic activity to various extents and exhibited resistance to encephalomyocarditis virus (EMCV) but not to vesicular stomatitis virus replication. The specific resistance to EMCV can be attributed to 2-5A synthetase. o isso Academic

Press.

Inc.

INTRODUCTION

cDNA had 1.4 kb or shorter inserts and corresponded to the prototype L3 clone. The L3 cDNA detects two transcripts of 1.7 and 2.3 kb in the cytoplasmic RNA of IFN-treated L-929 cells. This clone encodes the 43kDa enzyme which is recognized by the anti-peptide B antibody mentioned above and shows 2-5A synthetase activity. NIH-3T3 cells were transfected with L3 cDNA and expressed constitutively 2-5A synthetase. The expression of this enzyme (even in the absence of IFN treatment) correlates with resistance of the cells to encephalomyocarditis virus (EMCV) but not to vesicular stomatitis virus (VSV) replication.

The molecular mechanisms by which interferons (IFNs) inhibit replication of many viruses, suppress cell growth, and produce regulatory effects on cellular physiology are not yet fully elucitated. The 2’-5’oligo A (2-5A) synthetase is a well-studied IFN-induced protein. This enzyme is able to reproduce in vitro the IFNinduced inhibition of mRNA translation. The 2-5A synthetase comprises multiple enzyme forms with different requirements for double-stranded RNA activation and different intracellular localization on ribosomes, membranes, cell sap, and nucleus (Chebath et al., 1987b). 2-5A synthetase transcripts of 1.6, 1.8, and 3.6 kb are expressed in various human cell lines (Merlin et a/., 1983). The various enzyme forms (100, 67, 46, and40kDainhumancellsand110,70,and43kDain mouse cells (Chebath et a/., 198713)) are recognized by the same antibodies directed against a peptide (peptide B) common to the two human cloned enzymes encoded by 1.6-and 1.8-kb mRNAs (Benech eta/., 1985; Chebath et al., 1987b). In this paper we describe the isolation of a murine full-length cDNA (L3) of 2-5A synthetase. We screened a cDNA library made from total poly(A)+ RNA of mouse L-929 cells treated with fibroblast IFN using as a probe the human 2-5A synthetase cDNA El 8 (Merlin et al., 1983). Most clones cross-hybridizing to the human

MATERIALS Cell cultures

0042-6822/90

$3.00

Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

METHODS

and IFN

L-929 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 5% fetal calf serum (FCS) and antibiotics; NIH-3T3 cells were grown in Dulbecco’s modified minimum essential medium (DMEM) with 10% calf serum (CS). NIH-3T3 cells transfected with a construct containing the neomycin resistance gene were kept in medium supplemented with 0.5 mg/ml of G418, a neomycin analog (GIBCO). Mouse fibroblast IFN was prepared and partially purified to 1 O7 units/mg of protein as described (Coccia et a/., 1988). mRNA

Sequence data from this article have been deposited Bank Data Libraries under Accession No. M33863. ’ To whom requests for reprints should be addressed.

AND

with the Gen-

isolation

and cDNA

cloning

Poly(A)+ RNA was prepared and purified by the guanidine/cesium chloride method (Glisin et a/., 1974) fol228

MOUSE

2-5A

SYNTHETASE

lowed by separation on an oligo(dT)-cellulose column (yield: 0.4 mg per 10’ cells). The cDNA was prepared from oligo(dT)-primed poly(A)+ RNA of IFN-treated L-929 cells. The RNA-DNA hybrid was converted to double-stranded cDNA by treatment with RNase H and DNA polymerase I of fscherichia co/i (Gubler and Hoffman, 1983). The cDNA was methylated with EcoRl methylase and ligated to synthetic EcoRl linkers, and oligomeric terminal linkers were cleaved with EcoRI. The excess linkers were removed by gel filtration through a Bio-Gel A-50 column and the cDNA was ligated to Xgtl 1 arms (Young and Davis, 1985) previously digested with EcoRl and dephosphorylated with calf intestinal alkaline phosphatase (Promega). After in vitro packaging, the library of 1 O6 plaque forming units (PFU) was amplified in f. co/i Y 1088, a /aclq strain that represses transcription of the P-galactosidase-cDNA fusion protein. cDNA

library screening

The Xgtl 1 cDNA library was screened by cross-hybridizing to human full-length cDNA E-18 (Benech et a/., 1985). The insert corresponding to the human 1.8kb mRNA was purified by agarose gel electrophoresis and nick-translated (Rigby et a/., 1977). Plaques were repeatedly picked from g-cm’ plates (3 X 1 O5 phages) and small DNA preparations were analyzed by restriction mapping using routine procedures (Maniatis et a/., 1982). Several Xgtl 1 cDNA clones hybridizing to E-18 cDNA were isolated and the longest one was cut with IZcoRI and the insert subcloned into pGEM-3 (Promega) to obtain pGEM-L3.

AND

RESISTANCE

TO

229

EMCV

[35S]dATP was used as the radionucleotide mmol, 10 mCi/ml).

(>400

Ci/

pLj-L, construction The pLj vector (Fig. 3) consists of a Moloney murine leukemia virus (M-MuLV) transcriptional unit derived from an integrated M-MuLV provirus and pBR322 sequences necessary for the propagation of the vector DNA in E. co/i (Mulligan, 1983; Bernstein et a/., 1985; Temin, 1986). L3 cDNA was inserted into the BarnHI site in the correct orientation under the transcriptional control of the 5’ LTR. The pLj vector containing L3 was termed pLj-L3. Transfection

of NIH-3T3

ceils

Cells (5 X 105) were grown in a 90-mm dish for 18 hr. The culture medium was then replaced with 10 ml fresh medium and 4 hr later 10 pg of calcium phospate precipitated (plj or plj-L,) DNA was added (Wigler et al., 1979). After 20 hr at 37” the DNA-containing medium was replaced with fresh medium and 24 hr later the cells were trypsinized and seeded into new dishes at a splitting ratio of 1:8. After an additional 24 hr, the medium was replaced with medium containing 0.5 mg/ ml G418 (Geneticin, GIBCO) and was changed every 2-3 days. G418-resistant clones (1 O-40 per dish) appeared after 2 weeks. Northern

blot analysis

After T7 RNA polymerase transcription of plasmid (1 pg) linearized by Xbal, the corresponding RNA (0.05 pg) was translated in a 25-~1 reaction mixture containing 6 ~1 of reticulocyte lysate pretreated with micrococcal nuclease and 6 &i of [35S]methionine (>800 Ci/mmol, Amersham). 2-5A synthetase activity was tested on 5~1 aliquots of in vitro translation reactions as described below.

Poly(A)+ RNA (10 pg), extracted as before, was electrophoresed through denaturing 1.2% agarose gels containing 0.4 M formaldehyde, transferred onto nitrocellulose filters, and hybridized to nick-translated 32Plabeled (1.5 X 1O6 cpm/ml) murine L3 or pSV2-neo probes. The hybridization conditions were 50 mM sodium phosphate (pH 7.0) 50%formamide, 5X SSC, 4X Denhardt’s solution, 0.1% SDS, 200 pg/ml salmon sperm DNA at 42” for 20 hr. The filters were washed twice in 1X SSC and 0.1% SDS for 30 min at room temperature, twice in 0.1 X SSC, 0.1% SDS for 30 min at 42” and exposed to X-ray film.

Nucleotide

2-5A synthetase

cDNA

transcription-translation

sequence

determination

pGEM-L3 cDNA clone was sequenced using the Promega Gem/Seq Sequencing system. Deletions of the L3 insert were generated from 5’ and 3’ by using exonuclease III (Stratagene). The SP6 promoter and T7 promoter primers were annealed to the alkali-denaturated plasmids for priming chain termination dideoxy sequencing by Klenow or reverse transcriptase enzymes.

assay

The enzyme from cell extracts was bound to poly(rl).(rC)-agarose beads (PL Biochemicals) and incubated for 16 hr at 30” with 2.5 mM [a-3*P]ATP (300 mCi/ mmol, Amersham) (Chebath et a/., 1987b). The reaction mixture treated with alkaline phosphatase was electrophoresed and radioactivity incorporated into (A2’p),A cores was measured.

230

COCCIA ET AL. AL &a1 &a1

IA IA EcoRI

EcoRI QCII \

PVUII

5’

3

I

-

OSKb

t CCAGGCtGCG AGACCCAGGA ACCTCCAGAC TTAGC~GA GCACGGACTC ACCAGCATCC CAGCCTGGAC GCTGGACAAG TTCATACAGG ATTACCTCCT 101 TCCCGACACC ACCTTTGGTGCTGATGTCAA ATCAGCCGTC AATCTCGTGT GTGATTTCCTGAAGGAGAGA TGCTTCCAAG GTGCTGCCCACCCAGTGAGG 201 GTCTCCMGC TGGTGAAGGGTGGCTCCTCA GGCAMGGCA CCACACTCAA GGGCAGGTCAGACGCTGACC TGGTGCTCTTCCTTMCAAT CTCACCAGCT 301 TTGAGGATCA GTTAAACCGA CGGGGAGAGT TCATCAACGA AAttAAGAAA CAGCTGTACG AGGTTCAGCA TGAGAGACGTTTTAGAGTCAAGtltGAGGt A01 CCAGACTTCA TGGTGGCCCA ACGCCCGGTC TCTGAGCTTC AAGCTGAGCG CCCCCCATCt GCATCAGGAG GTGGACTTTGATGTGCTGCCAGCCTTTGAT 501 GTCCTGGGTCATGTTAATAC TTCCAGCAAG CCTGATCCCA GAATCTATGC CATCCTCATC GAGGAATGTACCTCCCTGGG GAAGGATGGCGAGTTCTCTA 601 CCTGCTTCAC CCAGCTCCAG CGGAACTTCC TGAAGCAGCG CCCAACCAAG CTGAAGAGTC TCATCCGCCT GGTCAAGCAC TGGTACCAAC TGTGTAAGGA 701 GAAGCTGGGGAAGCCATTGC CTCCACAGTA CGCCCTAGAG TTGCTCACTC TCTTTGCCTGGGAACAAGGG AATGCATGTT ATGAGTTCAA CACAGCCCAG 801 GGCTTCCGGA CCGTCTTGGA ACTGGTCATC AATTATCAGC ATCTTCGAAT CTACTGGACA AAGTATTATG ACTTTCAACA CCAGGAGGTC TCCAAATACC 901 TGCACAGACA GCTCAGAAAA GCCAGGCCTG TGATCCTGGA CCCAGCTGAC CCAACAGGW ATGTGGCCGG TGCGAACCCA GAGGGCTG~ GGCGGTTGGC to01 TGAAGAGGCT GATGTGTGGCTATGGTACCC A T G T T T T A T TAAAAAGGATG GTTCCCGAGT GAGCTCCTGG GATGTGCCGA CGGTGGTTCCTGTACCTTTT 1101 GAGCAGGTAG AAGAGAACTG GACATGTATC CTGCTCTQG CACAGCAGCA CCTGCCCAGG AGACTGCTGG TCAGCGCCAT TTGCTGCTCTGCTGCAGGCC 1201 CATGACCCAG TGAGGGAGGGCCCCACCTGG CATCAGACTC CGTGCTTCTGATGCCTGCCA GCCATGTTTGACTCCTGTCC AATCACAGCC AGCCTTCCTC 1301 AACAGATTCA GAAGGAGAGGAAAGAACACA CGCTTGGTGTCCATCTGTCC ACCTGTTGGA AGGTTCTGTC TGACAAAGTC TGATCAACAA TAMECACAG 1401 CAGGTGCCGT C(A)n

FIG. 1. Structure (A) and nucleotide sequence (6) of murine L, cDNA. The isolated L3 cDNA was subcloned in pGEM-3 and the cDNA insert was sequenced as described under Materials and Methods, The major restriction sites are indicated in the figure. The initiation codon, termination codon. and polyadenylation signal are underlined.

RESULTS Isolation

of 2-5A synthetase

cDNA clones

L-929 cells treated for 20 hr with 200 U/ml of murine fibroblast IFN were used to prepare the cDNA libraries since these cells markedly express a 1.7-kb mRNA that hybridized with the human probe. Several mouse cDNA libraries were then prepared by using Xgtl 1 as cloning vector and screened using the 3’P-labeled human cDNA (E-l 8) (Merlin et a/., 1983). A clone containing an insert about 1.4 kb long (L3) (Fig. IA) was picked from the various clones that hybridized strongly to the 32P-labeled human 2-5A synthetase probe. Most of the isolated clones had 1.4 kb or shorter inserts that corresponded to the same prototype LB. The major restriction sites of L3 are shown in Fig. 1A. L3 cDNA detects an abundant transcript of 1.7 kb and also one of mZ.3 kb (data not shown) in the cytoplasmic RNA of IFNtreated L-929 cells. Nucleotide sequence and transcription-translation assay of 2-5A synthetase L3 cDNA Mouse 2-5A synthetase L3 cDNA was subcloned into the EcoRl site of the multiple cloning site of pGEM3. The nucleotide sequence of the L3 cDNA insert is shown in Fig, 16. The sequence consists of 1400 nucleotides (and includes 20 bp of the 5’-untranslated region). A plausible initiation codon, the termination co-

don, and the poiyadenylation signal are underlined. The sequence of this murine cDNA was found to be the same as that determined by lchii (Ichii et al., 1986). The nucleotide sequence of the L3 clone shows 60% homology to the human cDNA El 8 (Merlin ef al., 1983) that encodes the 46-kDa 2-5A synthetase. The pGEM-L3 transcription-translation product (see Materials and Methods) labeled with [35S]methionine in reticulocyte lysates was analyzed by gel electrophoresis. A clearly identifiable product migrated as a protein of 43 kDa (Fig. 2, lane 3f which was not detectable in the control assay (Fig. 2, lane 2). This 43-kDa protein was recognized in a Western blot (data not shown) by antibodies against peptide B of the human 2-5A synthetase (Chebath et al., 1987b). In addition, the enzymatic assay performed on 5 ~1 of in vitro translation reaction demonstrated 2-5A synthetase activity (12,000 cpm of 32P-labeled oiigomers compared to 900 cpm in the control assay). Construction and properties of the pLj-L, vector and its transfection into murine NIH-3T3 cells (Fig. 3) We transfected murine NIH-3T3 cells with pLj or pljL3 by calcium phosphate precipitation (Wigler et a/., 1979). After 2 weeks of culture in 0.5 mg/ml G418, we selected permanently transformed cells by virtue of their resistance to G4 18.

MOUSE

2-5A

SYNTHETASE

AND

RESISTANCE

231

TO EMCV

4

100, 92.5,

12

3

4

4.7 Kb

5

FIG. 4. Northern blot of RNA extracted from NIH-4A2 (lane l), -5H3 (lane 2) -6G2 (lane 3) -6C3 (lane 4) and -1 F3 (lane 5) cell clones. RNA was extracted and analyzed as described under Materials and Methods. The indicated band is the expected 4.7.kb transcript. 46,

12

3

FIG. 2. Polyacrylamide gel electrophoresis of the [%]methioninelabeled translation products in a rabbit reticulocyte lysate. Ten percent SDS-polyacrylamide gel electrophoresis of the [%]methionine-labeled translatron products. The product of clone L3 is indicated in the autoradiogram (arrow). Lane 1, molecular weight markers; lane 2. control, no exogenous RNA; lane 3, RNA from pGEM-L, linearized by Xbal. Molecular weights (kDa) are indicated on the left.

The G418-resistant clones were analyzed by Southern and Northern blots to check the integration and transcription of the vector (data not shown) and by enzymatic assay to detect the presence of 2-5A synthetase activity. Figure 4 shows Northern blot analysis of

clones transfected with pLj-L, (NIH-1 F3, -6G2, -4A2, -6C3) or only with the pLj vector (NIH-5H3). The hybridization was performed with 32P-labeled L3 cDNA. Most of the analyzed clones showed the 4.7-kb mRNA corresponding to the recombinant retroviral genome encoding L3 and G418 resistance that hybridized strongly to L3 cDNA, while mRNAs from control cells (cells transfected with pLj alone) gave no signal (Fig. 4, lane 2). 2-5A synthetase enzymatic activity was about 20fold above the basal level in one clone (NIH-1 F3; Table l), whereas in the majority of clones expression of 25A synthetase activity was very low. The enzymatic activity observed in the NIH-1 F3 clone was equivalent to that of control cells (NIH-5H3) treated with 200 U/ml of fibroblast IFN for 20 hr (Table 1) and correlates with accumulation of mRNA that hybridized to L3 cDNA. Cell clones cultured for more than 4 months in G418containing medium showed relatively constant levels of 2-5A synthetase expression.

EMCV (but not VSV) replication is inhibited 3T3 clones expressing 2-5A synthetase

Polyoma

Early

Region

FIG. 3. Structure of the pLj-L, vector. For a description see Materials and Methods. The BarnHI restriction endonuclease cleavage site is indicated. L3 cDNA IS inserted at this sate under the transcriptional control of the 5’ LTR.

in NIH-

To evaluate the ability of transfected cells to replicate lytic viruses, we infected cells with EMC virus under one-step growth cycle conditions. Cells were infected at a multiplicity of 4 to obtain infection of more than 95% of the cells. Seven hours later the supernatant fluids were collected for evaluation of EMC virus yields by plaque assay on L-929 cell confluent monolayers. The two cell clones that expressed the highest levels of 25A synthetase (NIH-1 F3 and -6G2) showed 1.4 and 1.1 logs of reduction, respectively, of the yield with respect to that obtained from NIH-5H3 cells transfected with pLj (control cells). EMC virus yields obtained from four different NIH-3T3 cell clones transfected with pLj and 10 cell clones transfected with pLj-L, but not expressing 2-5A synthetase enzymatic activity averaged 8.7 f 3 X 1O7 (mean + a) PFW5 X 1O5 cells, indicating that the reduction of virus yield observed in NIH-lF3 and -6G2 cell clones represented the acquisition of an antiviral state that correlated with the expression of 2-5A synthetase enzymatic activity. To increase the reduction of virus yield and further verify a bona fide antiviral

232

COCCIA TABLE

REDUCTION OF EMC

VIRUS AND VW

ET AL. 1

YIELDS AND 2-5A SYNTHETASE ACTIVITY IN NIH-3T3

CELL CLONES TRANSFECTED WITH 2-5A SYNTHETASE L3 cDNA

A log PFU/5 X 1 O5 ceW EMCV Cell clones

m.0.i.

NIH-5H3 NIH-5H3 + IFN-(Y@ 200 U/ml NIH-1 F3 NIH-6G2 NIH-4A2 NIH-6C3 a Logs of EMCV/5 X NIH-5H3 is virus yields,

4

0 2.0 1.4 1.1

0.4 0.2

2-5A synthetase activity

vsv m.0.i.

1 Oe4

m.0.i.

4

cpm L3*P] 2-5A

0 2.6

0 2.5

387 9954

2.6 2.2

0.01 -0.5

8678 3200

0.1

0.1 N.D.

418 310

0.5

reduction of viruses yields compared to NIH-5H3 cell clone values: 6.3 X 1 O7 PFU of EMCV/5 X 1 O5 cells (m.o.i. 4), 4 X 1 O7 PFU of 1 O5 cells (m.o.i. 1 O-“), 4 X 10’ PFU of VSV/5 X 1 O5 cells (m.o.i. 4). NIH-1 F3, -4A2, -6G2, and -6C3 are pLj-L3 transfected clones; a plj-transfected control clone. Cells were seeded at 2.5 X 1 O5 cells/plate and infected with EMCV or VSV after 40 hr. To evaluate supernatants were collected at 7 hr (m.o.i. 4) or 24 hr (m.o.i. 1 O-4) after EMCV infection and 8 hr after VSV infection.

state, we performed experiments that permitted multiple cycles of viral multiplication. Cells (1 06) were infected with at a very low MOI (1 Om4) and supernatants were collected after more than three cycles of multiplication (24 hr). Under these experimental conditions EMC virus yields of NIH-lF3 and -6G2 dropped to values comparable to those obtained with control cells (NIH-5H3) treated with 200 U/ml of fibroblast IFN (Table 1). Interestingly, when the same cell clones were infected with VSV at MOI 4, no reduction in virus yield was observed in the transfected clones, whereas 2.5 log reduction was observed in NIH-5H3 control cells pretreated with murine fibroblast IFN (Table 1). DISCUSSION IFN treatment induces resistance to virus replication. There are several unanswered questions concerning the mechanisms that mediate this effect. More than 20 proteins are induced by IFNs including the 2-5A synthetase and the elF-2 protein kinase (Lengyel, 1982; Revel and Chebath, 1986). Much indirect evidence (Baglioni and Nilsen, 1983), including studies with cell lines that are partially responsive to IFNs (Epstein eta/., 1981; Affabris eta/., 1983; Romeo eta/., 1985; Sen and Herz, 1983; Lewis, 1988; Kumar et a/., 1988), indicates that these enzymatic pathways are involved in the establishment of the antiviral state. However, it is not clear which pathway plays a dominant role in preventing the replication of various types of viruses. The availability of full-length cDNAs clones that encode IFN-induced proteins may be useful for probing their roles in IFN action. The experiments described in this report were designed to investigate the role of the murine 2-5A synthetase in the antiviral state induced by IFNs. We

cloned murine 2-5A synthetase cDNA from a library of IFN-treated L-929 cells screened with a human 2-5A synthetase cDNA probe. The isolated full-size L3 clone is 1.4 kb long, shares 60% homology with the human 1.8-kb mRNA, and recognizes two transcripts of 1.7 and 2.3 kb in IFN-treated mouse cells. By transcriptiontranslation experiments we found that L3 encodes a 43kDa protein with the expected 2-5Asynthetase activity. NIH-3T3 cells were transfected with L3 cDNA under the control of a constitutive promoter. In the transfected cell clones, where 2-5A synthetase is accumulated in the absence of IFN treatment, inhibition of EMC virus replication was observed. In our experimental system the constitutive expression of this enzyme did not however protect cells against VSV replication, thus confirming that 2-5A synthetase is unable to prevent VSV replication (Chebath et a/., 1987a). In accord with data obtained in IFN-sensitive and -resistant cell variants on inhibition of replication of EMC virus (Lebleu and Content, 1982; Kumar et al., 1988) or VSV (Nilsen et al., 1980; Romeo et a/., 1985), it is apparent that the anti-VSV action of IFN can be dissociated from its antiEMC virus action. In fact, the 2-5A synthetase pathway seems to be directly involved in the inhibition of EMC virus but not of VSV replication. ACKNOWLEDGMENTS This study was supported by grants from the Italy-USA Program on Therapy of Tumors, Minister0 Pubblica Istruzione, Associazione ltaliana Ricerca sul Cancro and Consiglio Nazionale delle RicercheProgetto Finalizzato “Oncologia” N. 88.00487.44. The editorial assistance of Ms. Sabrina Tocchio is gratefully acknowledged.

REFERENCES AFFABRIS, E., ROMEO, G.. BELARDELLI, F., JEMMA, C., MECHTI, N., GRESSER, I., and ROSSI, G. B. (1983). 2-5A synthetase activity does not increase in interferon-resistant Friend leukemia cell variants

MOUSE

2-5A

SYNTHETASE

treated with al/3 interferon despite the presence of high-affinity interferon receptor sites. Virology 125,508-512. BAGLIONI, C., and NILSEN. T. W. (1983). Mechanisms of antiviral action of interferon. In “Interferon” (I. Gresser, Ed.), Vol. 5, pp. 2342. Academic Press, New York. BENECH, P., MORY, Y., REVEL, M., and CHEBATH. J. (1985). Structure of two forms of the interferon-Induced (2’.5’) oligo A synthetase of human cells based on cDNAs and gene sequences. EMBO /. 4, 2249-2256. BERNSTEIN, A., BERGER, S., HUSZAR, D., and DICK, J. (1985). Gene transfer with retrovirus vectors. In “Genetic Engineering: Principles and Methods” (J. K. Setlow and A. Hollaender, Eds.), Vol. 7, pp. 235-261. Plenum, New York. CHEBATH. J.. BENECH, P.. REVEL, M., and VIGNERON. M. (1987a). Constitutive expressjon of (2’.5’) oligo A synthetase confers resistance to picornavirus infection. Nature (Londort~330, 587-588. CHEBATH, J., BENECH, P., HOVANESSIAN, A., GALABRU, J., and REVEL, M. (1987b). Four different forms of interferon-induced 2’,5’-oligo(A) synthetase identified by immunoblotting in human cells. J. Viol. Chem. 262,3852-3857. COCCIA, E. M., FEDERICO, M., ROMEO, G., AFFABRIS, E., COFANO, F., and ROSSI, G. B. (1988). Interferon+a/p and y resistant Friend cell variants exhibiting receptor sites for interferons but no induction of Z-5A synthetase and 67K protein kinase. /. interferon Res. 8, 113-127. EPSTEIN, D. A., CZARNIECKI. C. W., JACOBSEN, H., FRIEDMAN, R. M.. and PANET, A. (1981). A mouse cell line which is unprotected by interferon against lflic virus infection lacks ribonuclease F activity. Eur. J. Biochem. 118, 9- 15. GLISIN, V., CRKVENJAKOV, R., and BYUS, C. (1974). Ribonucleic acid Isolated by cesium chloride centrifugation. Biochemistry 13, 2633-2636. GUELER, E.. and HOFFMAN, B. J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25,263-269. ICHII, Y., FUKUNAGA, R., SHIOJIRI, S., and SOKAWA, Y. (1986). Mouse 2-5A synthetase cDNA: Nucleotide sequence and comparison to human 2-5Asynthetase. NucleicAcids Res. 14, 10,l 17. KUMAR, R., CHOUBEY, D., LENGYEL, P.. and SEN. G. C. (1988). Studies on the role of the 2’.5’-oligoadenylate synthetase-RNase L pathway in beta interferon-mediated inhibition of encephalomyocarditis virus replication. /. Viral. 68, 3 175-3181. LEBLEU, B., and CONTENT, J. (1982). Mechanisms of interferon action: Biochemical and genetlc approaches. In “Interferon” (I. Gresser, Ed.), Vol. 4. pp. 47-94. Academic Press, New York.

AND

RESISTANCE

TO EMCV

233

LENGYEL, P. (1982). Biochemistry of interferons and their actions. Annu. Rev. Biochem. 51,251-282. LEWIS, J. A. (1988). Induction of an antiviral state by interferon in the absence of elevated levels of 2’.5’.oligo(A) synthetase and elF-2 kinase. Virology 162, 118-127. MANIATIS, T., FRITSCH, P., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. MERLIN, G., CHEBATH, J., BENECH, P., METZ. R., and REVEL, M. (1983). Molecular cloning and sequence of partial cDNA for interferon-induced (2’.5’)oligo(A) synthetase mRNA from human cells. froc. Nat/. Acad. Sci. USA 80,4904-4908. MULLIGAN, R. C. (1983). Construction of highly transmissible mammalian cloning vehicles derived from murine retroviruses. In “Experimental Manipulation of Gene Expression” (M. Inouye, Ed.), pp. 155-l 73. Academic Press, New York. NILSEN, T. W., WOOD, D. L., and BAGLIONI, C. (1980). Virus-specific effects of interferon in embryonal carcinoma cells. Nature 286, 178-180. REVEL, M., and CHEBATH, J. (1986). Interferon-activated genes. Trends Biochem. Sci. 11, 166- 170. RIGEY. P., DIECKMAN, M., RHODES, C., and BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. /. MO/. Biol. 113, 237-251. ROMEO, G., AFFABRIS, E., FEDERICO, M., MECHTI. N , COCCIA, E. M., JEMMA, C., and ROSSI, G. B. (1985). Establishment of the antiviral state in a/((1 interferon-resistant Friend cells treated with 7 interferon: InductIon of 67K protein kinase activity in absence of detectable 2-5A synthetase. J. Biol. Chem. 260. 3833-3838. SEN, G. C., and HERZ, R. E. (1983). Differential antiviral effects of interferon in three murine cell lines. /. Viral. 45, 1017- 1027. TEMIN, H. M. (1986). Retrovirus vectors for gene transfer: Efficient integration into and expression of exogenous DNA in vertebrate cell genomes. In “Gene Transfer” (R. Kucherlapati, Ed.), pp. 149187. Plenum, New York WIGLER, M., SWEET, R., SIM, G. K., WOLD, B., PELLICER, A., Lacy. E., MANIA% T., SILVERSTEIN, S., and AXEL, R. (1979). Transformation of mammalian cells with genes from procaryotes and eucaryotes. Proc. Nat/. Acad. Sci. USA 77, 3570.-3587. YOUNG, R., and DAVIS, R. (1985). lmmunoscreentng Xgtl 1 recombinant DNA expressjon libraries. In “Genetic Engineering: Principles and Methods” (J. K. Setlow and A. Hollaender, Eds.). Vol. 7, pp. 29-41. Plenum, New York.