Characterization of a vaccinia virus-encoded double-stranded RNA-binding protein that may be involved in inhibition of the double-stranded rna-dependent protein kinase

VIROLOGY

185206-216

(1991)

Characterization of a Vaccinia Virus-Encoded Double-Stranded RNA-Binding Protein That May Be Involved in Inhibition of the Double-Stranded RNA-Dependent Protein Kinase JULIA C. WATSON,’

HWAI-WEN CHANG, AND BERTRAM L. JACOBS*

Department of Microbiology,

Arizona State University, Tempe, AZ 85287-2701

Received May 2 1, 199 1; accepted July 22, 199 1 The work described in this article identifies a vaccinia virus-encoded protein that may be involved in inhibition of the interferon-induced, double-stranded RNA-dependent protein kinase. Extracts prepared from vaccinia virus (WR strain)infected cells contain an inhibitor of this kinase. Inhibition was reduced in extracts from which dsRNA-binding proteins had been removed by preadsorption to poly(rl) - poly(rC)-Sepharose, suggesting that a dsRNA-binding protein may be involved in kinase inhibition. A single major virus-specific polypeptide of M, = 25,000 (~25) bound to the poly(rl) . poly(rC)-Sepharose. ~25 was synthesized in e coupled in vitro transcription/translation system programmed with vaccinia cores, indicating that it is a vaccinia-encoded protein. Synthesis of ~25 was detected at early times, by 2 hr post infection, peaked at 5 hours postinfection, and decreased during the late phase of virus replication. In the presence of cytosine arebinoside ~25 synthesis did not decrease at late times postinfection. Kinase inhibitory activity accumulated with similar kinetics to ~25, both in the presence and absence of cytosine arabinoside. Kinase inhibitory activity copurified with ~25, through gel filtration, and Cibacron blue-affinity chromatography. Removal of ~25 by precipitation with antiserum to ~25 decreased kinase inhibitory activity in extracts prepared from vaccinia virus-infected cells. These results suggest that ~25 may be necessary for the specific kinase inhibitory activity detected in vaccinia virus-infected cells. Q 1991 Academic Press. Inc.

INTRODUCTION

unit of elF-2 blocks the GDP-GTP exchange cycle which is required for elF-2 function (Safer, 1983). This block in elF-2 function results in a halt of initiation of protein synthesis, including viral protein synthesis. A number of viruses have recently been shown to produce inhibitors of the P,/elF-2 kinase. Such inhibitors may enable these viruses to synthesize proteins even in the presence of potentially active kinase and to escape the antiviral effects of interferon. Several mechanisms of inhibition have been reported. The adenovirus inhibitor is a small RNA called VA I RNA (Kitajewski et al., 1986), that appears to bind to kinase, but not activate kinase (Katze et a/., 1987). The VA I RNA is apparently involved in the resistance of adenovirus to IFN treatment, since mutants lacking the VA I gene are IFN sensitive (Kitajewski et al., 1986). Influenza virus infection also induces the synthesis of an inhibitor of the P,/elF-2 kinase. This inhibitor is thought to be a cellular protein that inhibits P, autophosphorylation (Katze et a/., 1988, Lee et al,, 1990). Reovirus-infected cells also contain an inhibitor of the P,/elF-2 kinase (Imani and Jacobs, 1988). The virus-encoded dsRNAbinding protein, ~3, is both necessary and sufficient for the kinase inhibitory activity found in extracts prepared from reovirus-infected cells (Imani and Jacobs, 1988). ~3 is thought to function by binding to dsRNA, thereby reducing the effective concentration available for kinase activation (Imani and Jacobs, 1988). Histone pro-

Treatment of cells with interferon3 leads to the production of several enzymes that together can inhibit the synthesis of proteins and can lead to the establishment of an antiviral state (reviewed by Lengyel, 1982). One of these IFN-induced enzymes is a protein kinase that can autophosphorylate a M, = 66,000 enzyme subunit, P,, and can phosphorylate the protein synthesis initiation factor, elF-2, on its (Ysubunit. This kinase (P,/elF-2 kinase) is induced in an inactive form. Kinase becomes activated after interaction with dsRNA (Ziberstein et a/., 1976). Since dsRNA is produced during many viral infections (Boone el a/., 1979), it may act as an indicator of viral infection. Once activated, kinase can phosphorylate elF-2. Phosphorylation of the (Ysub’ Present address: Department of Medicine, Division of Infectious Diseases, Stanford University Medical School, Stanford, CA 94305. ’ To whom reprint requests should be addressed. 3 Abbreviations used: ara-C, cytosine arabinoside; BCIP, 5-bromo4-chloro-indolyl phosphate; BMV. brome mosaic virus; dsRNA, double-stranded RNA; DlT, dithiothreitol; elF-2a, the small subunit of eukaryotic protein synthesis initiation factor 2; hpi. hours postinfection; MEM, minimal essential medium; MOI, multiplicity of infection; NBT, nitrotetrazolium blue; P,, the M, = 67,000 subunit of the dsRNA-dependent protein kinase; PBS, phosphate-buffered saline; pfu. plaque forming unit; s-MEM, minimal essential medium for suspension; VA I RNA, sdenovirus-associated RNA I. 0042-6822191

$3.00

Copyright Q 1991 by Academic Press. Inc. All rights of reproduction in any form reservad.

206

VACCINIA

VIRUS

&RNA-BINDING

teins, which can bind to dsRNA as well as DNA, have kinase inhibitory activity, similar in nature to the reovirus a3 protein (Jacobs and Imani, 1988). Vaccinia virus has been shown to produce an inhibitor of the P, /elF-2 kinase (Paez and Esteban, 1984a, Rice and Kerr, 1984, Whitaker-Dowling and Youngner, 1983). The inhibitor has been reported to be proteinaceous and to interact noncatalytically with dsRNA (Whitaker-Dowling and Youngner, 1984). In extracts prepared from vaccinia-infected cells, the dsRNA concentration required for kinase activation is shifted to higher levels compared to extracts prepared from uninfected cells (Whitaker-Dowling and Youngner, 1984; this manuscript). On the basis of these observations, it seemed plausible that the vaccinia virus-mediated inhibition of the P, /elF-2 kinase could be due to a dsRNAbinding protein, similar to histone proteins and the reovirus ~3 protein. The experiments presented in this manuscript describe a vaccinia virus-encoded M, = 25,000 dsRNA-binding protein (~25) that is synthesized with similar kinetics to kinase inhibitory activity and copurifies through several fractionation steps with kinase inhibitory activity. Precipitation with antiserum that recognizes ~25 reduced kinase inhibitory activity. These results suggest that ~25 may be a necessary component of the vaccinia virus inhibitor of the dsRNAdependent P,/elF-2 kinase. MATERIALS

AND METHODS

Cell growth Mouse L cells were grown as monolayers at 37” in MEM supplemented with 5% fetal calf serum and 50 pg/ml gentamycin sulfate. HeLa cells were grown in suspension in s-MEM supplemented with 5% fetal calf serum and 50 pglml gentamycin sulfate; BSC-40 cells were grown as monolayers in MEM supplemented with 10% heat-inactivated fetal calf serum and 50 pg/ ml gentamycin sulfate. Virus stocks and virus infections Stocks of vaccinia virus (strain WR) were propagated in HeLa cells in suspension and titered in BSC40 cells according to the protocol of Mackett et al. (1985). Viral infections were carried out as follows. Virus stock was diluted to give the indicated MOI in MEM containing 1% fetal calf serum and 50 fig/ml gentamycin sulfate. Confluent monolayers of L cells in loo-mm plates were washed with PBS, and then infected with a total volume of 0.5 ml of diluted virus. Where mock infection is indicated, 0.5 ml of MEM without virus was added. After incubation at 37” for 60 min to allow virus adsorption, plates were overlaid with MEM containing 5% fe-

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tal calf serum and 50 &ml gentamycin sulfate, then incubated at 37”. Where treatment with ara c is indicated, the drug was added at a final concentration of 40 pg/ml to the virus inoculum and to the cell growth medium. Unless otherwise specified, infections were at a multiplicity of 5 pfu/cell, in the absence of ara c, and extracts were prepared at 4 hpi. Preparation

of cytoplasrnic

extracts

Cell monolayers were washed with cold isotonic buffer (35 mMTris-HCI, pH 6.8‘146 m&I N&I, 11 mM glucose), collected by scraping into this buffer, then harvested by centrifugation at 1000 g, Cells were lysed in a NP-40 lysis buffer(20 mM HEPES, pH 7.5,120 mM KCI, 5 mM MgCI,, 1 mM DTT, 10% glycerol, and 0.5% NP-40) using approximately 100 rlll0’ cells. Nuclei were removed by centrifugation at 10,000 g for 5 min at 4”. The resulting cytoplasmic extracts were stored at -80”. Pl/elF-2

kinase inhibitory

activity

Extracts prepared from vaccinia-infected cells were mixed with an equal volume of extract prepared from IFN-treated, uninfected L cells as a source of kinase. The mixed samples (10 ~1) were adjusted to 100 PM [T~~P]ATP (1 Ci/mmol), 20 mM HEPES, pH 7.5, 90 mM KCI, 5 mlW MgOAc, 1 mM DlT, and the indicated concentration of reovirus dsRNA in a final volume of 25 ~1, and the reaction mixture was incubated for 15 min at 30”. Reactions were quenched by the addition of an equal volume of 2x SDS/PAGE sample buffer. Samples were heated at 100” for 3 min then separated by SDS/PAGE (1970). Autoradiography was used to visualize phosphorylated proteins. Phosphorytation of the M, = 66,000 protein P, was used as an indication of kinase activity. Kinase inhibitory activity was determined by comparing the concentration of dsRNA required to activate kinase in various cell extracts. Alternatively, kinase inhibitory activity was assayed by monitoring the reduction of incorporation of 32P into protein P, , at a single, limiting concentration of dsRNA. Since the concentration of dsRNA necessary to overcome kinase inhibitory activity was proportional to the amount of inhibitor present, these two assays yielded similar results (data not shown). Poly(rI) . poly(rC)-Sepharose

binding

Poly(rl) . poly(rC)-Sepharose (Pharmacia) was washed three times in Buffer A (20 mM HEPES, pH 7.5,50 m/l/l KCI, 10% glycerol, 5 mM MgOAc, 1 mM DTT, 1 m/v9 benzamidine). Resin was preincubated for 30 min with bovine serum albumin at a final concentration of 20 mg/ml to block nonspecific binding sites, then washed

WATSON, CHANG, AND JACOBS

208

again in Buffer A. Cell extracts (50-1.00 ~1 of detergent lysate) were adjusted to a final concentration of 1 mM NAD+ (Wells eta/., 1984) and 1 mM benzamidine, then mixed with 25 ~1 of washed resin. In experiments to test the effect of competing soluble nucleic acids, the indicated concentration of nucleic acid was added to the cell extract immediately prior to mixing extract and resin. Resin and cell extracts were incubated on ice for 45 min. In experiments to determine kinase inhibitory activity after removal of dsRNA binding proteins, the resin was pelleted by centrifugation, the supernatant was removed and treated with 50 pglml micrococcal nuclease and 5 mM CaCI, to digest any contaminating dsRNA. Nuclease was inactivated by the addition of 20 mM EGTA and 1.2 mM thymidine-3’,5’-bis-phosphate. The supernatant solutions were then assayed for kinase inhibitory activity as described above. In experiments to identify dsRNA binding proteins, the resin was washed three times in Buffer A. dsRNA-binding proteins were removed from the resin by adding an equal volume of 2X SDS/PAGE sample buffer and heating samples at 100” for 3 min. Proteins were separated by SDS/PAGE. Rabbit antibodies

to p25

Electrophoretically purified ~25 was used to immunize a rabbit by the method of Boulard and Lecroisey (1982). Extracts of ara c-treated vaccinia-infected L cells prepared at 20 hpi were used as a source of ~25. The protein was isolated by poly(rl). poly(rC)-Sepharose adsorption and SDS/PAGE. The gel was soaked in 100 mM KCI at 4” to visualize protein bands. The M, = 25,000 band was excised, and the gel slice was washed in water, crushed in PBS, and mixed with an equal volume of Freund’s complete (for primary immunization), or incomplete (for secondary immunizations) adjuvant. This mixture was used for subcutaneous immunization of a rabbit. A total of approximately 3 pg protein was used in each immunization. Western

blot analysis

Proteins were separated by SDS/PAGE, then electrophoretically transferred to nitrocellulose in 25 mlMTris, 192 mlM glycine, 20% methanol, pH 8.6, at 4’, 100 V for 60 min. The blot was incubated overnight at room temperature in 10 mMTris-HCI pH 8.0, 150 mM NaCI, 10% Tween 20 (TBST) containing 1% gelatin (TBSTgelatin) to block nonspecific binding sites. TBST-gelatin was removed and replaced with crude anti-p25 serum (or preimmune serum, as indicated) diluted 1: 1,000 in TBST. After 30 min incubation at room temperature, the primary antibody solution was removed and the blot was washed 3 times, 5 min per wash, in

TBST at room temperature. The final wash was replaced with goat anti-rabbit IgG-alkaline phosphatase conjugate diluted 1 :l,OOO in TBST. Incubation in this secondary antibody solution was for 30 min at room temperature, followed by equilibration in 100 mMTrisHCI pH 9.2, 100 mM NaCI, 5 mM MgCI, (AP buffer). Bound secondary antibody-alkaline phosphatase conjugate was detected by a color development reaction using NBT and BCIP (Mierendorf et al., 1987). Stocks of the color development substrates were prepared immediately prior to use: NBT, 50 mg/ml in 70% dimethylformamide and BCIP, 50 mg/ml in 100% dimethylformamide. One hundred and thirty-two microliters of NBT stock and 66 /II of BCIP stock were combined with 20 ml of AP buffer. The nitrocellulose filter blot was placed in 20 ml of the NBT-BCIP solution in AP buffer. The reaction solution was covered with foil to protect light-sensitive reagents and incubated at room temperature for 20 min. Color reaction was stopped by washing the nitrocellulose in water. Immune

precipitation

For radioimmune precipitation, 5 ~1 of preimmune or immune serum was incubated for 1 hr on ice with 20 ~1 of extract from [35S]methionine labeled, vaccinia virusinfected cells. Prewashed, fixed S. aureus cells (70 J, Boehringer) were added and incubation was continued for 1 hr on ice. The bacterial cells were pelleted in a microfuge at 4” and washed three times in RIPA buffer (Springer, 1990) and once in 0.1 M Tris-Cl, pH 7.4. Bound proteins were eluted by boiling in SDS-PAGE lysis buffer for 3 min. For removal of kinase inhibitory activity, 30 ~1 of serum was incubated on ice for 1 hr with 150 ~1of fixed S. aureus cells that had been prewashed in buffer A. The antibody-coated cells were washed twice with Buffer A and then incubated for 1 hr at 4’ with 50 ,uI of extract from ara-C-treated, vaccinia virus-infected cells. The bacterial cells were removed and the supernatant solution was incubated with fresh antibody coated bacterial cells overnight at 4”. The bacterial cells were removed by centrifugation and the supernatant solutions were assayed for kinase inhibitory activity. ln vitro transcription/translation Coupled transcription and translation reactions were carried out using vaccinia cores, as described by Cooper and Moss (1978). Vaccinia virions were purified from crude virus stocks by centrifugation at 25,000 g through a continuous 15-40% sucrose gradient. Vaccinia cores were released from purified virions by incubating 50 A,,, of the purified virions in 0.5% NP-

VACCINIA VIRUS dsRNA-BINDING

40, 10 ml\/l DTT, 50 mhllTris-HCI, pH 8.5, at 37” for 30 min. The vaccinia cores were collected by centrifugation at 10,000 g for 1 min at 4”. For coupled in vitro transcription and translation, reactions contained the following components: 0.1 A,,dml vaccinia cores, 2 mM ATP, 0.2 mM CTP, UTP, and GTP, 20 PM amino acids minus methionine, 70% rabbit reticulocy-te lysate (Promega), and 1 mCi/ml [35S]methionine. Newly synthesized dsRNA binding proteins were identified by poly(rl) . poly(rC)-Sepharose adsorption followed by SDS/PAGE and autoradiography or Western blot analysis. [35S]methionine-labeling cells

of vaccinia virus-infected

Confluent monolayers of L cells were infected with vaccinia virus at an MOI of 40 pfu/ml and maintained in the presence or absence of ara c. Cells were incubated at 37” until 30 min prior to the designated time point post infection, when medium was removed and replaced with methionine-free medium (MEM, methionine-free, serum-free, with or without ara c). After 30 min incubation, medium was removed and replaced with labeling medium (MEM, methionine-free, serumfree, containing 40 &i/ml [35S]methionine at 1000 Gil mmol), with or without ara c. Plates were incubated 30 min in labeling medium. Lysates were prepared as described above. Lysates of mock infected cells were prepared in the same manner at 2 hr after mock infection. Protein purification To prepare cytoplasmic extracts for chromatography, mouse L cells were grown in suspension to high density, then infected with vaccinia. Cells were concentrated by centrifugation and resuspended into l/lOOth volume of s-MEM containing 1% fetal calf serum. Virus was added at an MOI of 5 pfu/cell. Ara c was added to a final concentration of 40 pg/ml; cell suspension was incubated 1 hrat 37”. The cell suspension was diluted back to its original volume in s-MEM containing 5% fetal calf serum and 40 pg/ml ara c. Infected cells were incubated 24 hr at 37”. At 24 hpi, cells were harvested by centrifugation at 2000 g and washed 3 times in ice cold isotonic buffer (35 mMTrisHCI, pH 6.8, 146 mM NaCI, 11 mM glucose). Cells were resuspended in 2.5 packed cell volumes of hypotonic buffer (10 mM HEPES, pH 7.5, 1.5 m/M MgOAc, 1 mlM DlT, 1 mM benzamidine, 10 mM KCI), swollen on ice for 15 min, then broken by dounce homogenization. The suspension was brought to 20 mM HEPES, pH 7.5, 5 mlW MgOAc, 1 mM DTT, 1 m/M benzamidine, and 150 m/W KCI and centrifuged at 5000 g for 10 min

PROTEIN

209

at 4” in a Sorvall SS-34 rotor. The supernatant solution was centrifuged at 30,000 g for 20 min at 4” in an SS-34 rotor. The supernatant solution from this second centrifugation was centrifuged at 1OO,UOOg for 2.5 hr at 4” in a Beckman Ti 50.1 rotor, to pellet ribosomes. The supernatant solution (S-100) was stored at -20” and used for further purification of ~25. A 2 ml vol of the S-l 00 fraction from 5 X 1O* vaccinia-infected cells was applied to a loo-ml bed volume column of Sephacryl S-200 which had been equilibrated in 20 rnM HEPES, pH 7.5, 5 mM MgOAc, 1 mNI DlT, 1 mM benzamidine, 10% glycerol, and 150 mM KCI (gel filtration buffer). Column flow rate was 0.5 ml/ min; 1-ml fractions were collected and stored at -20”. The gel filtration column was standardized separately with a mixture of proteins containing 0.5 mg each: alcohol dehydrogenase (M, = 150,000), bovine serum albumin (M, = 66,000), ovalbumin (M, = 45,000) cytochrome c (M, = 12,500) and either chymotrypsinogen (M, = 25,000) or carbonic anhydrase (M, = 29,000). Fractions were assayed directly for p25 by Western blot analysis, and for kinase inhibitory activity by addition of aliquots of the fractions to standard kinase assays containing 0.3 pg/ml reovirus dsRNA. Following gel filtration, fractions containing ~25 and kinase inhibitory activity (fractions 27-37) were pooled and loaded onto a l-ml column of Cibacron Blue-Sepharose (Pierce) packed for use with the Pharmacia FPLC system and equilibrated in gel fittration buffer. The column was run at 4” at a flow rate of 0.5 ml/mini l-ml fractions were collected. The column was washed in lo-column volumes of gel filtration buffer. Bound proteins were eluted in an increasing KCI gradient from 150 mM to 2 Mover 20 ml. Aliquots of column fractions (50 ~1) were desalted by centrifugation through a 0.5 ml Sephadex G-25 that had been saturated with ovalbumin and equilibrated in buffer A containing 0.1% Triton X-l 00. Desalted fractions were assayed for kinase inhibitory activity and ~25 as described for fractions from the gel filtration column.

RESULTS

An increased concentration of &&MA ia required for activation of the P,/elF-2 kinaae in ~#@acts prepared from vaccinia virus-infected ceNs The effect of vaccinia infection on P,IelF-2 kinase activation was analyzed by comparing the dsRNA concentration required for kinase activation in extracts from mock-infected and vaccinia-infected cells. P,/elF2 kinase activity was assayed by monitoring transfer of

210

WATSON,

CHANG.

AND

JACOBS

Kinase inhibitory activity in extracts from vacciniainfected cells is reduced by adsorption to poly(rl) . poly(rC)-Sepharose To determine if the vaccinia-induced inhibitor of the P,/elF-2 kinase might be a dsRNA-binding protein, such proteins were partially removed by adsorbing extracts to poly(rl) apoly(rC)-Sepharose. The supernatant solutions from these binding reactions were assayed for P,/elF-2 kinase inhibitory activity. The results of these assays are shown in Fig. 2. Fig. 2A shows P,/ elF-2 kinase activation in extracts prepared from vaccinia-infected cells without preliminary dsRNA-binding. Fig. 26 shows activation in extracts in which dsRNAbinding proteins had been removed by a preliminary poly(rl) s poly(rC)-Sepharose adsorption step. Activation of P,/elF-2 kinase was detected at a 1O-fold lower

FIG. 1. Inhibition of P,/elF-2 kinase activity by extracts from vaccinia-infected cells. Monolayers of mouse L cells were infected with vaccinia, or mock-infected. NP-40 extracts were prepared as described under Materials and Methods. Extracts from vaccinia-infected or mock-infected cells were mixed with extracts from IFNtreated cells as the donor of kinase, then assayed for kinase activity as described under Materials and Methods. Phosphorylated proteins were separated by SDS/PAGE. Autoradiograms of the dried gels are shown. In both panels, the kinase reaction mixtures contained the following concentrations of added reovirus dsRNA: lane A, no dsRNA; lane B, 0.01 pglml; lane C, 0.1 &ml; lane D, 1.0 rglml; lane E, 10.0 pg/ml. Arrow denotes the P, enzymes subunit phosphorylated by active P,/elF-2 kinase.

radioactive phosphate from [r3*P]ATP to the M, = 66,000 substrate of the P,/elF-2 kinase. Autoradiography of SDS/PAGE separated proteins was used to visualize phosphorylated proteins. Fig. 1 shows that when extract from mock-infected cells was incubated with extract from interferon-treated cells as a source of kinase, the P,/elF-2 kinase was activated at a dsRNA concentration of 0.01 pg/ml (Fig. 1A, lane 6). When extract prepared from vaccinia virus-infected cells was incubated with extract prepared from interferontreated cells, 1 @g/ml dsRNA was required to reach a comparable level of kinase activation (Fig. 1 B, lane D). These results confirm that extracts prepared from vaccinia virus-infected cells contain an inhibitor of the dsRNA-dependent P,/elF-2 kinase (Paez and Esteban, 1984a, Rice and Kerr, 1984, Whitaker-Dowling and Youngner, 1983) and that inhibitory activity can be overcome by increasing the concentration of dsRNA added to reactions (Whitaker-Dowling and Youngner, 1984).

a

b

c

d

e

B

FIG. 2. Partial removal of P,/elF-2 kinase inhibitory activity from extracts of vaccinia-infected cells by adsorption to poly(rl). poly(rC)-Sepharose. Monolayers of mouse L cells were infected with vaccinia and maintained in the presence of ara c. NP-40 cytoplasmic extracts were prepared at 20 hpi as described under Materials and Methods. Extracts were assayed for P,/elF-2 kinase activity as described under Materials and Methods, either without preadsorption to remove dsRNA-binding proteins (A), or after dsRNA-binding proteins were removed by incubating extracts with poly(rl). poly(rC)-Sepharose (B) as described under Materials and Methods. The supernatant solution, containing unbound proteins, was treated with micrococcal nuclease to remove small amounts of dsRNA that eluted from the resin, then assayed for P,/elF-2 kinase activity, as in Fig. 1. In both panels, the kinase reaction mixtures contained the following concentrations of added reovirus dsRNA: lane a, no dsRNA; lane b, 0.01 &ml; lane c, 0.1 *g/ml; lane d, 1 .O ag/ml; lane e, 10.0 pg/ml. Arrow denotes the P, enzymes subunit phosphorylated by active P,/elF-2 kinase.

VACCINIA VIRUS dsRNA-BINDING

PROTEIN

21i

resin in the presence of competing soluble singlestranded RNA (Fig. 4, lane C), double-stranded DNA (Fig. 4, lane E), and single-stranded DNA (Fig. 4, lane F). Soluble dsRNA could competiiively block binding of p25 to the dsRNA resin since the protein did not appear among the bound proteins when soluble dsRNA was added (Fig. 4, lane D). These results suggest that p25 binds specifically to dsRNA, and is not a general nucleic acid binding protein. Rabbit antibodies

FIG. 3. dsRNA-binding proteins in vaccinia-infected and mock-infected mouse L ceils. Monolayers of mouse L cells were infected with vaccinia virus (lane B) or mock-infected (lane A). NP-40 extracts were prepared as described under Materials and Methods. dsRNAbinding proteins in the extracts were isolated by adsorption to poly(rl). poly(rC)-Sepharose as described under Materials and Methods Proteins bound to the resin were eluted by boiling in SDS sampie buffer, then separated by SDS/PAGE and Coomassie stained. Arrow denotes vaccinia virus-specific, M, = 25,000, dsRNA-binding protein (~25).

to ~25

Electrophoretically purified ~25 was used to immunize a rabbit, as described under Materials and Methods. The crude antiserum was used in a Western blot analysis against total proteins in extracts prepared from uninfected and vaccinia-infected cells. Results are shown in Fig. 5. Immune serum reacted positively with a M, = 25,000 protein in extracts prepared from vaccinia-infected cells (Fig. 5, lane C), but bib not recognize any similar protein in the extracts prepared from uninfected cells (Fig. 5, lane D). Preimmune serum did not react with proteins in either cell extract (Fig. 5, lanes A, B). Serum from the immunized rabbit could also precipitate a M, = 25,000 protein from extract of

dsRNA concentration in the preadsorbed extract (Fig. 2B, lane d), than in untreated extract (Fig. 2A, lane e), indicating that when dsRNA-binding proteins were partially removed from extracts of vaccinia-infected cells, inhibition of the P,/elF-2 kinase was decreased. Vaccinia virus infection results in the appearance a M, = 25,000 protein, ~25, that binds specifically to dsRNA

of

In order to detect vaccinia-specific dsRNA-binding proteins, extracts from mock-infected and vaccinia-infected mouse L cells were incubated with poly(rl). poly(rC)-Sepharose. Bound proteins were eluted with SDS/PAGE sample buffer, separated by SDS/PAGE, and identified by Coomassie staining. Fig. 3 shows the results of this experiment. Extracts prepared from both mock-infected and vaccinia-infected cells contained proteins which could bind to dsRNA. However, only one major protein was present in extracts prepared from vaccinia-infected cells and not in extracts prepared from mock-infected cells. This protein can be seen in Fig. 3B and electrophoreses with M, = 25,000. We have designated this M, = 25,000, vaccinia virusinduced, dsRNA-binding protein as ~25. Fig. 4 shows the results of addition of completing soluble nucleic acids to binding reactions. Coomassiestained proteins remaining bound to the resin are shown. The ~25 protein remained bound to the dsRNA

A

B

C

D

E

I=

FIG. 4. p25 binding to poly(rl) . poly(rC)-Sepharose in the presence of competing soluble nucleic acids. Monolayers of mouse L cells were infected with vaccinia and maintained in the presence of ara c, and NP-40 extracts were prepared at 20 hpi, as described under Materials and Methods. Extracts were incubated with poly(rl). poly(rC)-Sepharose in the presence of competing soluble nucleic acids, as described under Materials and Methods. In each reaction, the bound dsRNA provided by the resin was 80 rg; in each reaction 100 pg of soluble competing nucleic acid was added. Proteins remaining bound to the resin were e&ted by boiling in SOS sample buffer, separated by SDS/PAGE, and Coomaaele stained. Lane A, chymotrypsinogen (M, = 25,000), as a molecular weight marker; lane B, no competing soluble nucleic acid. Soluble nucleic acids added: lane C, poly(rA) (single-stranded RNA); lane D, poly(rl). poly(rC) (dsRNA); lane E, calf thymus DNA (double-stranded DNA); lane F, denatured calf thymus DNA (single-stranded DNA).

212

WATSON. CHANG, AND JACOBS

A

C

B

D

Pre-imm dsRNA

130

-

+

A

B

Anti

+

D25

+ -R

CDEFGHI

JK

FIG. 7. Immune clearance of p25 and kinase inhibitory activity. Extract from araC-treated vaccinia virus-infected cells was incubated with either S. aureus-bound preimmune serum (lanes C-F), or S. aureus-bound anti-serum to p25 (lanes G-K). Bacterial cells were removed by centrifugation, and kinase inhibitory activity was assayed in the supernatant solutions in the presence of 0.3 ag/ml of reovirus dsRNA. The following amounts of extract were added to standard kinase inhibitory assays: lanes C and G, 0.1 pl; lanes D and f-f, 0.3 pl; lanes E and I, 1 .Opl; lanes F and J, 3.0 ~1; and lane K, 6.0 ~1. In each case extract from uninfected cells was added to bring the total volume of extract added to the assays to 6.0 ~1. Lanes A and B represent kinase assays in extract from interferon-treated cells in the absence (lane A) or presence (lane B) of 0.3 pg/ml of reovirus dsRNA.

39 27

FIG. 5. Western blot analysis of proteins from vaccinia-infected cells and uninfected cells. Monolayers of mouse L cells were infected with vaccinia, or left uninfected. NP-40 cytoplasmic extracts were prepared as described under Materials and Methods. Proteins were separated by SDS/PAGE, then transferred to nitrocellulose as described under Materials and Methods. Western blot analysis was carried out as described under Materials and Methods. Primary antibody was either serum from a rabbit immunized as described under Materials and Methods using electrophoretically purified ~25. or preimmune serum from the same animal. Lanes A and C show proteins from vaccinia-infected cells; lanes B and D show proteins from uninfected cells. Lanes A and B were incubated with preimmune serum; lanes C and D were incubated with immune serum.

vaccinia virus-infected cells labeled at early times postinfection with [35S]methionine (Fig. 6, lane B). To determine if p25 might be associated with the vaccinia virus-induced inhibitor of the P,/elF-2 kinase we assayed the supernatant solutions of immune precipitation reactions for kinase inhibitory activity. As shown in Fig. 7, extracts from which p25 had been removed by precipitation with antiserum made to p25 contained no detectible kinase inhibitory activity at the

Pre

Imm

II

4p25

A

B

FIG. 6. Radio-immune precipitation from extracts of vaccinia virusinfected cells. Extract from ara-C-treated, vaccinia virus-infected cells that had been labeled at 20 hpi, was incubated with either preimmune (lane A) or immune serum (lane B) from a rabbit that had been immunized with purified ~25. Complexes were washed in RIPA buffer, and bound proteins were eluted with SDS/PAGE sample buffer, resolved by SDS/PAGE, and visualized by autoradiography.

concentration of dsRNA used in these assays (0.3 pg/ ml) even when 6 ~1 of extract was added to the reactions (Fig. 7, lane K). As little as 1 ~1of extracts that had been preincubated with preimmune serum (Fig. 7, lane E) had detectable kinase inhibitory activity. Quantitative scanning densitometry of the gel in Fig. 7 indicated a 70-759’0 reduction of incorporation of 32Pi into P, by addition of 1 ~1 of extract adsorbed with preimmune serum and no reduction by addition of 6 ~1 of extract adsorbed with antiserum to ~25. p25 is a vaccinia-encoded

gene product

Although p25 was present in extracts from vacciniainfected cells and was not detectable in extracts from uninfected cells, the possibility remains that p25 could be a cell-encoded gene product that is induced during vaccinia infection. In order to determine whether p25 is a vaccinia gene product, coupled transcription and translation reactions programmed with vaccinia cores were performed. dsRNA-binding proteins were purified from the reaction mixture and separated by SDS/ PAGE. Fig. 8A shows that a M, = 25,000 dsRNA-binding protein was among the translation products synthesized in reactions containing vaccinia cores (Fig. 8A, lane c). This protein was not detected when vaccinia cores were omitted from the reaction (Fig. 8A, lane b), or when BMV RNA was used to program translation reactions (Fig. 8A, lane a). Rabbit polyclonal antiserum to p25 was used to probe the products of the in vitro transcription/translation reactions. As seen in Fig. 88, the M, = 25,000 translation product of vaccinia cores reacted with antibodies to p25 (Fig. 88, lane c), while BMV translation products did not (Fig. 8B, lane a). Vaccinia virus cores alone did not react positively with antibodies to p25 (data not shown). These results indicate

VACCINIA

be

dsRNA-BINDING

8

A

a

VIRUS

a

b

c

FIG. 8. dsRNA-binding proteins synthesized in a coupled in vitro transcription and translation reaction programmed with vaccinia cores Vaccinia cores were used to program a coupled transcription and translation reaction in a cell-free rabbit reticulocyte lysate in the presence of f6SImethionine. as described under Materials and Methods. Newly synthesized dsRNA-binding proteins were isolated and separated by poly(rl) . poly(rC)-Sepharose adsorption and SDS/ PAGE, as described under Materials and Methods. An autoradiogram of the gel is shown in (A). Lane a shows translation products from a reaction programmed with BMV RNA. Lane b shows translation products from a reaction with no further additions; lane c shows translation products from a reaction programmed with vaccinia cores (B) The results of a Western blot analysis of a duplicate gel, using rabbit anti-p25 serum as primary antibody. Molecular weight standards are indicated on the right.

that p.25 is a vaccinia gene product and is not a component of cores. p25 is an early vaccinia virus gene product In order to follow the synthesis of ~25 during the course of infection, cells were infected and maintained in the presence (Fig. 9A, lanes f-j), or absence (Fig. 9A, lanes a-e) of ara c. Newly synthesized proteins were labeled with [?S]methionine at 2,5,8, and 12 hr postin-

A

araC + araC -0 2 5 8 12 0 2 5 8 12 hpi

8

- araC

PROTEIN

213

fection. Late viral proteins are synthesized after DNA replication, which is blocked in the presence of ara c; thus no late viral proteins were synthesized in the extracts from ara c-treated cells (Fig. 9A, compare lanes e and j). Extracts from labeled celis were bound to poly(rl) . poly(rC)-Sepharose to enrich for ~25 (Fig. 96). In the absence of ara c (Fig. 9B, lanes a-e), synthesis of ~25 was detected at 2 hr postinfection, reached a peak at 5 hr, and decreased by 12 hr postinfection. In the presence of ara c (Fig. 9B, lanes f-j), ~25 was synthesized continuously through 12 hr postinfection. Similar results were obtained when p25 was isolated from extracts of vaccinia virus-infected untreated cells by precipitation with anti-p25 serum (Fig. SC). These results demonstrate that ~25 is an early protein, since it appeared in extracts prepared at 2 hr postinfection and was synthesized in the presence of ara c. In order to analyze the kinetics of accumulation of kinase inhibitory activity, extracts prepared at various times post infection were assayed for the concentration of dsRNA necessary to activate kinase. Fig. 10 shows these results. In the absence of ara c (solid circles) the peak of kinase inhibitory activity was detected at 5 hr postinfection, since at that time kinase activation required the highest concentration of added dsRNA. In the presence of ara c (open circles), kinase inhibitory activity increased through 12 hr postinfection. These results suggest that kinase inhibitory activity, like ~25, is an early vaccinia virus function, consistent with previously reported results (Whitaker-Dowling and Youngner, 1984). Kinase inhibitory

activity and ~25 co-purify

We have begun to purify both p25 and kinase inhibitory activity from the postribosomai supernatant frac-

+ araC

Ill 025812

0258Mhpi

abcde

fghi

C

j

g

4

115

a

b

c

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d

FIG. 9. Time course of synthesis of ~25 in vaccinia virus-infected mouse L cells. Cells were infected wrth vaccinia at a MOI of 40 pfu/ml, in the presence (lanes f-j) or absence of ara c (lanes a-e). At the designated time post infection, cells were labeled with [36S]methionine and MP-40 extracts were prepared as described under Materials and Methods. Extracts designated as 0 hpi were mock-infected, Newly synthesized proteins in these extracts were separated by SDS/PAGE and visualized by autoradiography (A). dsRNA-binding proteins in the extracts were isolated by adsorption to poly(rl). poly(rC)-Sepharose as described under Materials and Methods. Bound proteins were eluted by boiling in SDS sample buffer, separated by SDS/PAGE, and visualized by autoradiography (B). In (C). p25 was precipitated from extracts prepared from untreated, virus-infected cells with anti-serum to ~2.5, as described under Materials and Methods. Immune precipitated proterns were solubilized in SDS sample buffer, separated by SDS/PAGE, and visualized by autoradiography (C).

214

WATSON, CHANG, AND JACOBS

0

5

10

15

Hours poet infection FIG. 10. Accumulation of P,/elF-2 kinase inhibitory activity during vaccinia infection. Extracts from vaccinia-infected cells were prepared as described in Fig. 9 and assayed for P,/elF-2 kinase inhibitory activity, as described under Materials and Methods. Kinase inhibitory activity is expressed as dsRNA concentration required for detectable kinase activation.

tion (S-l 00) of ara-c-treated vaccinia virus-infected cells. The p25 and kinase inhibitory activity containing S-l 00 fraction was applied to a gel filtration column in a buffer containing 150 mM KCI. Column fractions were assayed for both kinase inhibitory activity and ~25. The results are shown in Fig. 1 1A. The bulk of the kinase inhibitory activity (open circles in Fig. 11A) eluted in fractions 27-37, with a native M, = 66,000. The bulk of the p25 (solid circles) eluted in these same fractions. The ~25 and kinase inhibitory activity-containing fractions from gel filtration were pooled and applied to the affinity resin Cibacron Blue-Sepharose. Western blot assays of desalted column fractions showed that p25 bound to this resin and eluted from the resin as a single peak (closed circles, Fig. 11 B), at a KCI concentration of approximately 1 M (peak at fraction 24). Kinase inhibitory activity (open circles) coeluted with ~25. DISCUSSION The antiviral effects of IFN have been correlated to two enzyme pathways: the 2’,5’-A synthetase-RNAse L system and the dsRNA-dependent P,/elF-2 kinase. Vaccinia infection appears to interfere with both the 2’,5’-A synthetase system (Paez and Esteban, 1984b; Rice et al., 1984) and the P,/elF-2 kinase (Paez and Esteban, 1984a; Rice and Kerr, 1984; Whitaker-Dow-

ling and Youngner, 1983). The work described in this article was initiated to better define the inhibitory effects of vaccinia infection on the IFN-induced P,/elF-2 kinase. Both of the IFN-induced antiviral pathways require interaction with dsRNA for enzyme activation. dsRNA binding proteins, such as histones and the reovirus a3 protein, have been shown to function as inhibitors of the P,/elF-2 kinase, presumably by competing with the kinase for binding to dsRNA (Jacobs and Imani, 1988; lmani and Jacobs, 1988; lmani and Jacobs, manuscript in preparation). The vaccinia-specific inhibitor of the PJelF-2 kinase has also been reported to be a protein that interacts in a non-catalytic manner with dsRNA (Whitaker-Dowling and Youngner, 1984). Therefore, it seemed likely that dsRNA-binding proteins produced during vaccinia infection could be potential kinase inhibitors. The work reported in this paper describes a vaccinia virus-encoded immediate early protein that binds specifically to dsRNA and may be involved in kinase inhibition. A single major dsRNA-binding protein (M, = 25,000) designated ~25, was detected in extracts prepared from vaccinia-infected cells, but not in extracts prepared from uninfected cells. Binding of this protein to poly(rl). poly(rC)-Sepharose could be efficiently competed with soluble dsRNA but not with single-stranded RNA, single-stranded DNA, or doublestranded DNA. p25 appears to be a virus-encoded polypeptide since it could be detected among the products of coupled in vitro transcription/translation reactions These results, along with the synthesis of p25 at early times postinfection suggest that p25 is an immediate early vaccinia virus protein. A second, smaller dsRNA-binding protein was also visible in some extracts prepared from vaccinia-infected cells. This protein may be a breakdown product of p25 since it reacted with antibodies to electrophoretically purified ~25. Several of our results suggest that ~25 is involved in and may be necessary for inhibition of the P,/elF-2 kinase. Two methods for the specific removal of p25 from extracts of vaccinia virus-infected cells also removed kinase inhibitory activity. Kinase inhibitory activity could be reduced either by adsorption of extracts with poly(rl). poly(rC)-Sepharose, or by adsorption of extracts with immobilized antibodies that recognize ~25. Kinase inhibitory activity and p25 copurified through gel filtration and Cibacron blue-Sepharose affinity chromatography. Both p25 and kinase inhibitory activity chromatographed through a column of Sephacryl S-200 with M, = 66,000. These results suggest that active ~25, capable of binding dsRNA, is either multimeric in nature, or complexed with some other

VACClNlA

VIRUS dsRNA-BINDING

A

0.7

1.4 -

150

66

2;

3.0

PROTEIN B

4!

Ii?% . 3 i.o2‘ . bm 0.6 3

.

1

0.6 -

b 2 !a

0.4 . 0.2 1 0

i

lb

15

2.0

Fraction

3;

4'0

4;

s-o-

#

Fraction

#

FIG. 11. Partial purification of P,/elF-2 kinase inhibitory activity and ~25. Protein ffOm the S-l 00 fraction of vaccinia virus-infected araC-treated cells was chromatographed through a column of Sephacryl S-200 (A). Individual fractions were assayed for kinase inhibitory a&v&y (open circles) in the presence of 0.3 fig of reovirus dsRNA/ml and for ~25 by Western blot analysis (solid circles). Fractions 27-37 were pooled and chromatographed through a column of Cibacron-blue-agarose (B). Individual fractions were assayed for kinase i‘nhibitory activity (open circles), ~25 (solid circles) and total protein (open squares) as in (A). Arrows denote the elution volume of standard proteins as described under Materials and Methods.

protein(s). Both kinase inhibitory activity and ~25 coeluted from a Cibacron blue-Sepharose affinity chromatography column at approximately 1 IM KCI, well included in the column, and well separated from the bulk of the protein loaded onto the column. The kinetics of synthesis of p25 are also consistent with its involvement in inhibition of the P, /elF-2 kinase. Synthesis of p25 reached a peak at 2-5 hr postinfection and decreased at late times postinfection (8-12 hpi). Kinase inhibitory activity also reached a peak at 2-5 hr postinfection, since the dsRNA concentration required for kinase activation was at its highest level at this time. In the presence of ara c, synthesis of ~25 and accumulation of kinase inhibitory activity continued to increase through 12 hr postinfection. Thus, both ~25 and kinase inhibitory activity appear to be early functions, whose accumulation could be superinduced by treatment with ara c. dsRNA is a potent activator of several of the enzymes that may be involved in establishing the IFN-induced antiviral state. Despite the fact that dsRNA is produced in detectable amounts in infected cells (Boone et al., 1979) replication of vaccinia virus is resistant to IFN treatment in some cells (Paez and Esteban, 1984a). This resistance may be due to the presence of inhibitors of several of the IFN-induced enzymes in vaccinia virus-infected cells. Vaccinia virus infection of non-IFN-treated BSC-40 cells induces a function, encoded by the ts22 gene, that prevents accumulation of 2’,5’-A. At restrictive temperature, late gene expression is halted in ts22-infected cells, con-

comitant with a buildup of active 2’,5’-A (Cohrs et al., 1989). The rs22 gene is transcribed at early a$ well as late times postinfection and codes for a protein of predicted molecular weight of approximately 56,000 (R. Condit, personal communication). In IFN-treated, vaccinia-infected CV-1 or L929 cells, 2,5-A accumulates to appreciable levels at late times postinfection, but there is a lag in activation of RNase L (Rice eta/,, 1984). Paez and Esteban (1984b) have reported the induction in vaccinia-infected cells of a phosphataae that could inactivate 2’,5’-A. As described previously (Paez and Esteban, 1984a; Rice and Kerr, 1984; Whitaker-Dowling and Youngner, 1983) and further characterized in this report, vaccinia virus-infected cells atso contain an inhibitor of the P,ielF-2 kinase. This inhibitor is an early viral function that may be the dsRNA-binding M, = 25,000 polypeptide that was described in this manuscript. Since the kinetics of synthesis of ~25 and kinase inhibitory activity are different from the kinetics of transcription of the ts22 gene, and since the molecular weights of ~25 and the ts22 gene product differ, it is likely that neither p25 nor the vaccinia virus-induced kinase inhibitor are encoded by the ts22 gene. It is at present unclear which of the vaccinia virus-encoded inhibitors of the IFN-induced enzymatic pathways is necessary to confer resistance to IFN treatment. It is conceivable that the vaccinia virus-encoded modulators of the IFN-induced enzymes may have effects on macromolecular synthesis, beyond their proposed effects on sensitivity to IFN treatment. While induced in cells by IFN treatment, most of the enzymes

216

WATSON. CHANG. AND JACOBS

postulated to be involved in the IFN-induced antiviral state are present at lower levels in non-IFN treated cells (Kimchi er al., 1979). In fact, phosphorylation of elF-2a! has been detected at late times postinfection in non-IFN-treated adenovirus-infected cells (O’Malley et a/., 1989), despite the synthesis of the viral P,/elF-2 kinase inhibitor, VA I RNA. It has been proposed that partial phosphotylation of elF-2 may allow for modulation of host protein synthesis in adenovirus-infected cells (O’Malley et a/., 1989). A similar phenomenon may be occurring during reovirus infection, since the virus-encoded kinase inhibitor, u3 protein has been implicated in the modulation of host protein synthesis (Sharpe and Fields, 1982). The vaccinia virus-encoded modulators of the IFN-induced enzymes may serve a similar function during vaccinia virus infection, either modulating hst protein synthesis, or being involved in the shift from early to late gene expression. ACKNOWLEDGMENTS We thank Dennis Hruby for providing BSC-40 cells, and Richard Condit for communication of results prior to publication. This work was supported in part by Grants BRSG 2 507 RR071 12. Division of Research Resources, National Institutes of Health; CA 48654, National Cancer Institute; and 8277-000000-l-O-YC-8357, Arizona Department of Health Services.

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KITAIEWSKI,J., SCHNEXIER,R. J., SAFER,B., MUNEMITSU,S. M., SAMUEL, C. E., THIMMAPPAYA.B., and SHENK, T. (1986). Adenovirus VA I RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced elF-2 kinase. Cell 45, 195200. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LEE, T. G., TOMITA, J., HOVANESSIAN,A. G., and KATZE,M. G. (1990). Purification and partial characterization of a cellular inhibitor of the interferon-induced protein kinase of M, 68,000 from influenza virus-infected cells. Proc. Nat/. Acad. Sci. USA 87, 6208-6212. LENGYEL, P. (1982). Biochemistry of interferons and their actions. Annu. Rev. Biochem. 51, 251-282. MACKEIT, M., SMITH, G. L., and Moss, B. (1985). The construction and characterization of vaccinia virus vectors expressing foreign genes. In “DNA Cloning. Vol. II. A Practical Approach.” (D. M. Glover, Ed.), pp. 191-211. IRL Press Ltd., Oxford, England. MIERENDORF,R. C., PERCY,C., and YOUNG, R. A. (1987). Gene isolation by screening X gtl 1 libraries with antibodies. In “Methods in Enzymology, Vol. 152. (S. L. Berger and A. R. Kimmel, Eds.), Vol. 2, pp. 458-469. Academic Press, San Diego. O’MALLEY, R. P., DUNCAN, R. F., HERSHEY,J. W. B., and MATHEWS, M. B. (1989). Modification of protein synthesis initiation factors and the shut-off of host protein synthesis in adenovirus-infected cells. virology 168, 112-l 18. PAEZ, E., and ESTEBAN,M. (1984a). Resistance of vaccinia virus to interferon is related to an interference phenomenon between the virus and the interferon system. Virology 134, 12-28. PAEZ, E., and ESTEBAN, M. (1984b). Nature and mode of action of vaccinia virus products that block activation of the interferon-mediated ppp(A2’p),A-synthetase. Virology 134, 29-39. RICE,A. P., ROBERTS,W. K., and KERR,I. M. (1984). 2-5A accumulates to high levels in interferon-treated, vaccinia virus-infected cells in the absence of any inhibition of virus replication. J. Viral. 50, 220228. RICE, A. P., and KERR, I. M. (1984). Interferon-mediated, doublestranded RNA-dependent protein kinase is inhibited in extracts from vaccinia virus-infected cells. 1. Viral. 50, 229-236. SAFER,B. (1983). 28 or not 28: Regulation of the catalytic utilization of elF-2. Cell 33, 7-8. SHARPE,A. H., and FIELDS,B. N. (1982). Reovirus inhibition of cellular RNA and protein synthesis: Role of the S4 gene. Urology 122, 381-391. SPRINGER,T. A. (1990). Purification of proteins by precipitation. In “Current Protocols in Molecular Biology, Volume 2” (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G.. Seidman, J. A. Smith, and K. Struhl, Eds.), Wiley, New York. WELLS, J. A., SWVRYD,E. A., and STARK, G. R. (1984). An improved method for purifying 2’,5’-oligoadenylate synthetases. J. Biol. Chem. 259, 1363-l 370. WHITAKER-DOWLING,P., and YOUNGNER,J. S. (1983). Vaccinia rescue of VSV from interferon-induced resistance: Reversal of translation block and inhibition of protein kinase activity. Virology 131, 128136. WHITAKER-DOWLING,P.. and YOUNGNER,1. S. (1984). Characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon-induced protein kinase. Virology 137, 171-181. ZILBERSTEIN,A., FEDERMAN,P., SHULMAN, L., and REVEL,M. (1976). Specific phosphorylation in vitro of a protein associated with riboSomes of interferon-treated mouse L cells. FEBS Letf. 68, 119124.