Pittsburgh, Pennsylvania 15261. Mouse L-cells infected with .... raphy-The isoelectric focusing method of O'Farrell (40) was modi- fied as described (39) for use ...
VOl. 264. No. 17. Issue of June 15, PP. 10321-10325,19S9 Printed in U.S . A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc
Vaccinia Specific Kinase Inhibitory FactorPrevents Translational Inhibition by Double-stranded RNA in Rabbit Reticulocyte Lysate* (Received for publication, December 7, 1988)
Giridhar R. Akkaraju, Patricia Whitaker-Dowling, Julius S. Youngner, and Rosemary JagusS From the Department of Microbiology, Biochemistry and Molecular Biology, Universityof Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Mouse L-cells infected with vaccinia virus produce a Refs. 2,5, and 6). This recycling mechanism involves guanine specific kinase inhibitory factor (SKIF) which inhibits nucleotide exchange on eIF-2, promoted by another initiation the activation of the interferon-induced, double- factor, eIF-2B (2, 5, 6). stranded (ds)RNA-dependent, eukaryotic initiation The reversible phosphorylation of eIF-2 seems to be an factor (eIF)-2a-specific protein kinase in L-cell ex- important general mechanism for regulating the rateof inititracts (Whitaker-Dowling, P., and Younger, J. S., ation. Kinases specific for the a subunit of eIF-2 have been (1984) Virology 137, 171). The effects of a partially identified in a wide variety of cells and tissues. These kinases purified preparation of SKIF have been examined in have been characterized most extensively in rabbit reticulocell-freeextracts of rabbit reticulocytes. Both the cytes which contain twoeIF-2a-specific kinases, a hemephosphorylation state of eIF-2 and protein synthetic sensitive kinase (eIF-2a-PKh)(7-9) and a dsRNA-dependent activity havebeen determined. SKIF inhibits the phos- protein kinase (eIF-Za-PKd,) (10-12). Although the hemephorylation of the a subunit of eIF-2 by dsRNA-de- sensitive and dsRNA-dependent kinase activities represent pendent eIF-2a-kinase in reticulocyte lysate, but does not affect phosphorylation of eIF-2 by the heme-sen- distinct protein species, activated by distinct signals, they a subunit(13). sitive kinase. In addition to its effects oneIF-2a-PKa. bothphosphorylatethesamesiteonthe Phosphorylation of eIF-2 by either kinase causes the inhibiactivity, SKIF prevents dsRNA-induced inhibition of protein synthesis in reticulocyte lysate. In contrast, tion of protein synthesis (1, 2). A dsRNA-dependent eIF-2a-specific protein kinase is inSKIF does not prevent the translational inhibition caused by hemin depletion. These data provide a direct duced in manycell types by interferons (14-16). Although not correlation between the effects of SKIF on eIF-2a phos- demonstrated definitively, this kinase shares many properties phorylation and on protein synthetic activity and dem- with, and is considered to be identical with, thereticulocyte onstrate the specificity of SKIF. The results also show eIF-2a-PKd, (reviewed in Ref. 17). Activation of this protein that SKIF does not abolish dsRNA sensitivity, but in- kinase, variously referred to in the literature P1 as (14, 18) or creases the concentration of dsRNA required to acti- P68protein (19) orPl/eIF-2kinase (ZO), occurs incells vate the kinase and phosphorylate eIF-2. infected by vesicular stomatitis virus (21, 22), encephalomyocarditis virus (23), and reovirus (24, 25) and leads to increases in the level of phosphorylation of eIF-2a (23, 26). This is thought toprovide a mechanism for the inhibition of In higher eukaryotes, acute regulation of protein synthetic protein synthesis whichoccurs as an antiviral response in rate in response to a wide variety of metabolic, autocrine, and hormonal factors occurs at the level of initiation of transla- interferon-treated, virus-infectedcells. Certain viruses, including vaccinia, adeno, and influenzaA, tion. Themajor control on the rate of initiation is at the level have evolved mechanisms that overcome the interferon-inof initiator tRNA binding to the small ribosomal subunit duced cellular defenses by inhibiting the activityof the inter(reviewed in Refs. 1 and 2). This is the step mediated by eukaryotic initiation factor 2, eIF-2l (3). eIF-2 is a trimer feron-induced eIF-2a-PKh(reviewed in Refs. 20 and 27). For composed of three disimilar subunits, designateda , /3,and y instance,inadenovirus-infected cells, VA1 RNAprevents (4). Its function is modulated by the regulatory a subunit (3), activation of a n eIF-%-specific kinase (28, 29). Similarly, the activityof which is inhibitedby phosphorylation (reviewed influenza A virus-infected cells contain an activity that prein Ref. 2). Phosphorylation of eIF-2 does not simply inactivate vents activation of eIF-2a-PKb (30). Thegrowth of vaccinia the factor, but perturbs the recycling mechanism (reviewed in virus is insensitiveto interferon in manycell types (31).Not only is vaccinia itself resistant to thedefenses of interferonviruses (21, * This work wassupported by National Institutes of Health Grants treated cells, but it is able to protect co-infecting GM-33905 (to R. J.) and AI-06264 (to J. S. Y.). The costs of publi- 22, 31, 32). The growth of vesicular stomatitis virus is norcation of this article were defrayed in part by the payment of page mally inhibited by interferon pretreatment of mouse L-cells, charges. This article must therefore be hereby marked “advertise- but this virusis able to grow in cells co-infected with vaccinia ment” in accordance with 18 U.S.C. Section 1734 solely to indicate virus (21, 22, 31). Co-infection with vaccinia also rescues the this fact. $ T o whom correspondence and reprint requests should be ad- growth of picornaviruses such as polio, mengo, and encephalomyocarditis virus in interferon-treatedcells (32-35). dressed. The abbreviations used are: eIF-2, eukaryotic initiation factor 2; The ability of vaccinia to grow in interferon-treated cells, eIF-2a-PKh,heme-sensitive kinase; eIF-2a-PK6, dsRNA-dependent as well as its ability to rescue the growth of other viruses, has protein kinase; SKIF, specific kinase inhibitory factor; ds, doublebeen correlated with the induction of a factor that prevents stranded; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l-propane- activation of eIF-Za-PKd, (21, 22, 35, 36). This activity repsulfonic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel resents a virus-encoded function (22, 36) and appears to bea protein that blocks both the autophosphorylation of eIF-2aelectrophoresis; VSIEF, vertical slab gel isoelectric focusing. 10321
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Effectof SKIF in Reticulocyte Lysate
PKd,, as well as the phosphorylation of eIF-Za in in vitro extracts (22,36).The activity has been termed specific kinase inhibitory factor or SKIF (22). This inhibitory protein factor appears early in infection (90 min), and itsinhibitory activity can be reversed by excess dsRNA (22). The data which suggest a relationship between the activity of the vaccinia-specific kinase inhibitory factor, SKIF, and the prevention of translational inhibition in vaccinia-infected, interferon-treated mouse L-cells, are only correlative. Translation of vaccinia-specific proteins is not reduced by prior interferon treatment, and this finding correlates with a lack of eIF-2a-PKds activity in extracts of interferon-treated vaccinia-infected L-cells (21). Similarly, co-infection with vaccinia allows the translation of vesicular stomatitis virus gene products in vesicular stomatitis virus-infected, interferontreated cells (21). Using partially purified SKIF and a cell-free translation system from rabbit reticulocytes, we have been able to document the effects of SKIF on both eIF-2aphosphorylation and translational activity directly. In addition, we have been able to show that SKIF inhibits the activity of eIF-2a-PKdS in reticulocyte lysate, but not thatof eIF-2a-PKh. MATERIALS AND METHODS
Reagents and Zsotopes"Poly(1:C) was obtained from the Antiviral Substances Collaborative Program of the National Institute of Allergy and Infectious Diseases. [14C]Valineand "'I-protein A (400 mCi/ mmol) were from DuPont-New England Nuclear. Rabbit anti-sheep antibodies (IgG, heavy plus light) were from Cappel. Immobilon-P was from Millipore. Cells and Viruses-Mouse L-cell monolayer cultures were grown and maintained as described (37). Stocks of vaccinia virus (Canada strain) were propagated and maintained as described (21). Production of Partially Purified SKZF-Monolayer cultures of Lcells in 100-mmtissue culture dishes were infected with vaccinia virus at a multiplicity of 10. Extracts were prepared at 5 h after infection. The cell monolayers were rinsed three times with phosphate-buffered saline, and the cells were permeabilized by the addition of 250 pl/ 100-mm dish of a buffer containing 20 mM HEPES, pH 7.5, 120 mM KCl, 5 mM MgCl,, 1 mM dithiothreitol, 10% (v/v) glycerol, and 0.5% (v/v) Nonidet P-40. After a 5-min incubation, the monolayer was scraped with a rubber policeman and transferred to a microfuge tube. The extracts were centrifuged in a microcentrifuge at 4°C a t 10,000 X g for 5 min. The supernatant (SlO) was collected and ultracentrifuged at 100,000 X g for 1 h in a Ti 60 rotor. The supernatant (SlOO) was fractionated with NH4S04, and thepelleted fraction containing SKIF (the 30-45% NH4S04 cut)was solubilized in 20 mM HEPES, pH 7.5,120 mM KCl, 5 mM MgCl,, 1 mM dithiothreitol, and 10% glycerol, at a concentration of 35 mg/ml and stored at -80°C. Rabbit ReticulocyteTranslation System-Rabbit reticulocyte lysate was prepared and used as described (38). Zn uitro translation reactions were performed with the addition of 100 mM KCl, 0.5 mM MgC12,150 p~ amino acids minus valine, ['4C]valine (50 pM, 120 mCi/mmol), 10 mM phosphocreatine, 2 units/mlcreatine phosphokinase, 20 p M hemin. The translation mix contained approximately 200 pmol of ribosomes/ml and exhibited a protein synthetic activity equivalent to 1pmol of globin/pmol of ribosome/min at 30°C. Determination of Phosphorylation State of eZF-2a in Reticulocyte Lysate-Reticulocyte lysate was incubated as described above in the presence or absence of SKIF and thepresence or absence of dsRNA. After incubation, 10-pl aliquots were added to 5 p1 of sodium fluoride (1M), towhich was added 185 pl of VSIEF sample buffer containing 9 M urea, 5% (v/v) P-mercaptoethanol, 2% (v/v) CHAPS (Fluka), 0.05% Pharmalytes, pH 4.5-5.4, 1%Pharmalytes, pH 5.5-6.1. The mixtures were vortexed, snap-frozen, and later subjected to vertical slab gel isoelectric focusing, VSIEF (39). Vertical Slab Gel Isoelectric Focusing/Transfer/Zmmunoautoradiography-The isoelectric focusing method of O'Farrell (40) was modified as described (39) for use in vertical slab gels. Reticulocyte samples werefocused through a narrow pH range of4.5-6.1 using narrow range Pharmalytes(PharmaciaLKB Biotechnology Inc.), in the presence of 9 M urea, 5% v/v p-mercaptoethanol, and 2% v/v CHAPS (Fluka). Proteins were electrophoresed at 2 mA/0.75-mm gel for 16
h, using reverse polarity, with 0.01 M glutamic acid at the anode and 0.05 M histidine at the cathode. Proteins were transferred to Immobilon-P at 60 V for 1 h in 250 mM glycine, 20 mM Tris, 20% (v/v) methanol, 0.01% (w/v) sodium dodecyl sulfate. After transfer, the nitrocellulose blots were blockedwith BLOTTO, 5% w/v nonfat dried milk, 0.01% Antifoam A (Sigma), 0.01% sodium azide in 50 mM Tris-HC1, pH 7.4,200 mM NaCl (41). This was followed by successive incubations with sheep anti-eIF-2, rabbit antisheep (Cappel), and 4pCi/ml of "'I-protein A (Du Pont-NewEngland Nuclear, specific activity 400 mCi/mmol). All antibody dilutions were made in BLOTTO. Immunoautoradiographs were made from the blots using Kodak XAR film. Polyacrylamide Gel Electrophoresis (SDS-PAGE)-Samples were analyzed on 15% SDS-polyacrylamide gels as described previously (42). RESULTSANDDISCUSSION
Effects of SKZF on Activity of the Reticulocyte Translation System-The effects of partially purified SKIF on translational inhibition by dsRNA and hemin deficiency are shown in Fig. 1. In the presence of hemin (20 PM),protein synthetic rates are linear and are equivalent to approximately 1 pmol of globin/pmol of 80 S equivalent/min. Partially purified SKIF (l%,-350 rg/ml, final concentration) has little effect on the rate of translation in the presence of hemin; protein synthetic activity is stimulated slightly. In the presence of dsRNA (150ng/ml), protein synthetic ratesproceed at control values for several minutes and then decline to reach a final rate between 5 and 10% of control values. This reflects the inactivation of eIF-2 which results from the activation of eIF2a-PKds (2). In the presence of partially purified SKIF, the inhibition of protein synthesiscaused by dsRNA is prevented. Since the SKIF preparations were only partially purified, it seemed possible that the prevention of dsRNA-induced
+SKIF
-S K I F + d r RNA
t SK I F
+ds RNA
-h -h
+SKIF
IO
20 T I M E (MIN.1
30
40
FIG. 1. Effect of SKIF on translational inhibition by dsRNA in a rabbit reticulocyte translation system. Cell-free extracts of rabbit reticulocyte lysate were incubated under standard conditions, as described under "Materials and Methods," and in the absence or presence of dsRNA (150 ng/ml), the absence or presence of hemin (20 p ~ )and , the absence or presence of SKIF (350 pglml). Protein synthetic activity was assayed by [14C]valineincorporation into trichloroacetic acid-precipitable material. A time course of incorporation is shown here.
Effect of SKIF in Reticulocyte Lysate inhibition could be caused by eIF-2 or eIF-2B contaminants in the SKIF preparation. Figs. 2, Panel A, shows an immunoautoradiograph of the partially purified SKIF preparation fractionated by SDS-PAGE and transferred to Immobilon-P. The blots were probed with sheep anti-eIF-2 and processed as described under “Materials and Methods.” Although the p and a subunits of eIF-2 are clearly visible in purified eIF-2 (Lane I), reticulocyte lysate (Lane 2), and L-cell lysate (Lane 4 ) , eIF-2 cannot be detected in the partially purified SKIF preparation (Lane 3 ) . This method is sensitive down to 0.10.2 pmol of eIF-2, demonstrating that the eIF-2 content of the SKIF preparation could not be more than 0.05 pmol/pl. This precludes the possibility that the restoration of protein synthetic activity by SKIF could be accounted for by eIF-2 contaminating the SKIF preparation: it has been demonstrated by a number of investigators that 10-30 pmol of eIF2 per 50 pl of translation mixture are needed to restore protein synthetic rates in dsRNA-treated or hemin-depleted reticulocyte cell-free translation systems (43, 44). The sheep antieIF-2 antiserum also contained antibodies reactive to the 82kDa subunit of eIF-2B, as shown in Fig. 2, Panel B. The 82kDa peptide of eIF-2B can be detected in purified eIF-2B (Lane I), reticulocyte lysate (Lane 2), and L-cell lysate (Lane 4 ) , but not in thepartially purified SKIF preparations (Lane 3). A demonstration of the specificity of SKIF can be seen by looking at its effect on the inhibition of translation thatoccurs during hemin deprivation. As seen in Fig. 1, incubation in the absence of hemin also leads to the inhibition of protein synthesis with biphasic kinetics. This inhibition results from phosphorylation of eIF-2a caused by activation of a heminsensitive kinase (14-16). In contrast to itseffects in dsRNAtreated lysate, SKIF does not prevent the inhibition of protein synthesis caused by hemin deprivation. In fact, the activity of the translationsystem is decreased slightly. Effects of SKIF on Phosphorylation of eIF-2a”Fig. 3 shows the effects of SKIF on the levels of phosphorylation of eIF2a. Lane 2 shows the phosphorylation state of the CY subunit in a control incubation in which protein synthetic rates are high. No phosphorylation of the CY subunit is observed. Lanes 1 and 3 show the phosphorylation of the CY subunit after incubation for 15 min in the absence of hemin or thepresence of dsRNA, respectively. In each case, approximately 30-35% of the pool of CY subunits are ina monophosphorylated form. After incubation in the presence of dsRNA and partially purified SKIF (Lane6), little phosphorylationof the CY subunit of eIF-2 is seen, indicating that SKIF inhibits activation of
A.
B. * ) . ~ 2 8
P
5
6
7
0 8
a 1
2
3
4
FIG. 2. eIF-2 and eIF-2B content of a preparation of SKIF. Samples were subjected to electrophoresis by SDS-PAGE (15% polyacrylamide), and the separated peptides were transferred electrophoretically to Immobilon-P as described under “Materials and Methods.’’ Immunoautographs were prepared as described under “Materials and Methods.” Panel A shows the content of a and @ subunits of eIF-2, Panel B shows the content of an 82-kDa subunit of eIF-2B. Lane I , 0.5 pmol of purified eIF-2; Lanes 2 and 6, 1 plof rabbit reticulocyte lysate; Lanes 3 and 7, 35 p g of SKIF; Lanes 4 and 8, 1 p1 of L-cell lysate; Lane 5,0.5 pmol of eIF-2B.
10323
-a
aP
1 2 3 4 5 6 FIG. 3. Effect of SKIF on phosphorylation of eIF-20 in a rabbit reticulocyte translation system. Cell-free extracts of rabbit reticulocyte lysate were incubated under standard conditions, as described under “Materials andMethods,” in the absence or presence of dsRNA (150 ng/ml), the absence or presence of hemin (20 p ~ ) and the absence or presence of SKIF (350 pg/ml). Samples were fractionated by VSIEF, transferred toImmobilon-P, and probed with sheep anti-eIF-2. Lane I , -h, -SKIF; Lane 2, +h, -SKIF Lane 3, +dsRNA, -SKIF Lane 4, -h, +SKIF; Lane 5 , +h, +SKIF; Lane 6, +dsRNA, +SKIF.
eIF-2a-PKd8and thusprevents inhibitionof protein synthesis. However, in keeping with the lack of effect of SKIF in preventing translational inhibition in heme-deficient reticulocyte lysate, SKIF does not prevent the phosphorylation of eIF-2a by eIF-2a-PKh (Lane4 ) . These findings confirm the specificity of SKIF and provide a direct demonstration that SKIF can prevent the reduction of translational activity by preventing phosphorylation of the N subunit of eIF-2. Effect of SKIF on Translation Response to a Range of dsRNA Concentrations-dsRNA only activates eIF-2a-PKd. over a fairly narrow range of concentrations (45, 46). The absolute concentrations of dsRNA required to activate eIF-2a-PKb vary with the type of dsRNA, its molecular weight, and the system underinvestigation. Fig. 4 shows the effects of increasing concentrations of dsRNA [poly(I:C)] on the translational activity of the reticulocyte translation system. dsRNA begins to inhibit at -50 ng/ml, gives maximum inhibition at 200400 ng/ml, and at higher concentrations loses its ability to inhibit translation. At concentrations higher than 2 pg/ml, dsRNA no longer causes significant translational inhibition. The mechanisms underlying this relationship between dsRNA concentration and activation of eIF-2a-PKh are not understood. It was of interest toknow whether SKIF prevents translational inhibition at any dsRNA concentration or displaces the concentration dependency. In in vitro protein kinase assays using extracts from interferon-treated L-cells, SKIF appears to preventactivation of eIF-2a-PKdS by interacting with dsRNA, and SKIF activity can be overcome by increasing concentrations of dsRNA (22). Similarly, the ratio of SKIF to ~ I F - ~ c Y - Paffects K ~ , the response of the kinase to a range of dsRNA concentrations in in vitro kinase assays. Fig. 4 shows the effects of increasing concentrations of dsRNA on protein synthesis in rabbit reticulocyte lysate in the presence of two concentrations of SKIF (0.5 and 1%,-175 and 350 pg/ml, respectively). In thepresence of each concentration of SKIF, a similar relationship between the rate of translation and dsRNAconcentration is observed to that occurring in the absence of SKIF, but in each case the bellshaped dose response curve is flattened and displaced to the right. In thepresence of 0.5%SKIF, protein syntheticactivity declines more slowly, reaching a low at 1 pg/ml. A t dsRNA concentrations higher than 2 pg/ml, protein synthetic rates rise again to control values. In the presence of 1% SKIF, the response curve to dsRNA is displaced even further to the right. Protein synthetic rate remains at control values until 600 ng/ml dsRNA, plateauing between 1 and 2 pg/ml, and rising again slowly to give control rates by 5 pg/ml. Effect of SKIF on eIF-2a Phosphorylation over a Range of dsRNA Concentrations-Fig. 5 shows that corresponding re-
,
Effect of SKIF in Reticulocyte Lysate
10324
FIG. 4. Effect of SKIF on translaof dsRNA in a tionalinhibition translation system from rabbit reticulocytes. Cell-free extracts of rabbit reticulocyte lysate were incubated under standard conditions, as described under “Materials andMethods,” in theabsence or presence of dsRNA (50 ng/ml-5 pg/ ml) and in the absence or presence of SKIF (175 or 350 pg/ml) for 30 min. Proteinsynthetic activity was assayed by [14C]valineincorporation into trichloroacetic acid-precipitable material. Data for the 30-mintime pointare shown here. 0, -SKIF; A, +SKIF (175 pglml); 0, +SKIF (350 pg/ml).
A
+dsRNA
+dsRNA
-h-
I
+ SKIF ( 0 . 5 % )
TABLE I
SKIF on phosphorylationof eZF-2a by dsRNA in a ” Effect of translation system from rabbit reticulocytes
+h
.”
”
Cell-free extracts of rabbit reticulocyte lysate were incubated under standard conditions, as described under “Materials and Methods,” in the absence or presence of dsRNA (50 ng/ml-5 pg/ml), and in the absence or presence of SKIF (350 pg/ml) for 30 min. Samples were fractionated by VSIEF, transferred to Immobilon-P, and probed with sheepanti-eIF-2.Immunoautographs were prepared as described ” under “Materials and Methods,” using preflashed film. Immunoautographs were scanned two-dimensionally on a Bio-Rad Model 620 +SKIF +SKIF video densitometer. The densitometric tracings were integrated and FIG. 5. Effect of SKIF on phosphorylation of eIF-2a by compressed into one-dimensional images and analyzed usingonedsRNA in a translation system from rabbit reticulocytes. Cell- dimensional Analyst software. free extracts of rabbit reticulocyte lysate were incubated under standHemin SKIF eIF-2a ard conditions, as described under “Materials and Methods,” in the dsRNA (20 ILM) (350 d m l ) phosphorylation absence or presence of dsRNA (50ng/ml-5 pg/ml) and in the absence % or presence of SKIF (350 pg/ml) for 30 min. Samples were fraction40 ated by VSIEF, transferred to Immobilon-P, and probed with sheep 3.5 anti-eIF-2. Lanes 1-7, -SKIF; Lanes 8-13, +SKIF (350 pg/ml); Lane 45 1, -hemin; Lanes 2-13, +hemin (20 p ~ ) Lanes ; 3 and 9, 100 ng/ml 54 dsRNA Lanes 4 and 10,200 ng/ml dsRNA; Lanes 5 and 1 1 , l pg/ml 26 dsRNA; Lanes 6 and 12, 2 pg/ml dsRNA; Lanes 7 and 13, 5 pg/ml 2.5 dsRNA. 4 2 lationships are found in the phosphorylation state of eIF-2a 3 over a range of dsRNA concentrations. In the absence of 8 SKIF, with increasing concentrations of dsRNA, phosphoryl50 50 ation of eIF-Sa is high at 100 and 200 pg/ml, is decreasing at 12 1 pg/ml, and is back to control values by 2 pg/ml. In the
YP L
I2 3 4 5 6 7 8 910111213
+ + + + + + + + + + + +
presence of 1%SKIF, phosphorylation of the CY subunit occurs only at higher dsRNAconcentrations, 1-2 pg/ml, and is prevented at dsRNA concentrations of 5 pg/ml. Table I shows the results of quantitating the change inphosphorylation state observed in Fig. 5. The relationship between dsRNA concentration and phosphorylation of eIF- CY corresponds to those observed between dsRNA concentration andtranslation in the presence or absence of SKIF. In addition, the effects of SKIF on the
response of both protein synthetic rate and eIF-2a phosphorylation to a range of dsRNA concentrations mimic the effects of SKIF on the response of eIF-2a-PKd, toa range of dsRNA concentrations in vitro (22). In both instances, the data support a direct interaction between SKIF and dsRNA rather than between SKIF and thekinase (22). There are now several examples of viruses which are able
Effect of SKIF in Reticulocyte Lysate to overcome the antiviral effects of interferon by preventing activation of eIF-2a-PKd, (reviewed in Refs. 27 and 28). The viral products responsible for the anti-interferon effects have in certain instances been identified and correlations made with the presence of the product and eIF-2a-PKh activity. Thisreport shows the direct manipulation of the rate of translation by the vaccinia anti-interferon product, SKIF. In addition,thesestudiesdemonstrateadirectcorrelation of the ability of the inhibitor to block eIF-2a phosphorylation and to interfere with the dsRNA-dependent inhibition of Protein synthesis. The ability of SKIFto inhibit the dsRNA-dependent protein kinase found in the reticulocyte is a further indication of the similarity of this kinase to the one induced by interferon and suggests that the reticulocyte translation system may be a useful system in which to investigate the mechanisms of action of other viral anti-interferon products. REFERENCES 1. Jagus, R., Anderson, W. F., and Safer, B. (1981) Proc. Nucleic Acids Res. Mol. Biol. 2 5 , 127-185 2. Pain, V. M. (1986) Biochem. J. 235,625-637 3. Safer, B., Adams, s.,Anderson, w. F., and Merrick, w. c. (1975) J. Biol. Chem. 250,9076-9082 4. Lloyd, M.A., Osborne, J. c., Jr., Safer, B., Powell, G. M., and Merrick, W. C. (1980) J. Biol. Chem. 2 5 5 , 1189-1193 5. Jagus, R. (1983) in P o s t t ~ a d a t ~ o n Modification al Of Proteins (Johnson, B. C., ed) pp. 159-180 6. Safer, B. (1983) Cell 33, 7-8 7. Trachsel, H., Ranu, R. s.9 and London, 1. M. (1979) Methods Enzymol. 6 0 , 485-495 8. Hunt, T. (1979) Miami Winter Symp. 321-346 9. Lundak, T. s.9 and TraWh, J. A. (1980) in Protein PhosPhoVhtion and Bio-Regulation (Thomas, G., Podesta, E. J., and Gordon, J., eds) pp. 154-161, S. Karger, Basel 10. Farrell, p., Balkow, K., Hunt, T., and Jackson, R. J. (1977) Cell 11,187-200 11. Ochoa, S. (1983) Arch. Biochem. Biophys. 2 2 3 , 325-334 12. Clemens, M. J., and McNurlan, M.A. (1985) Biochem. J . 2 2 6 , 345-360 13. Colthurst, D. R., Campbell, D.G., and Proud, C.G. (1987) Eur. J. Biochem. 1 6 6 , 357-363 14. Lebleu, B., Sen, G. C., Shaila, S., Cabrer, B., and Lengyel, P. (1976) Proc. Natl. Acad. Sci. U. S. A . 7 3 , 3107-3111 15. Kimchi, A., Zilberstein, A., Schmidt, A., Schulman, L., and Revel, M. (1979) J . Biol. Chem. 254,9846-9853 16. Roberts, W. K., Hovanessian, A., Brown, R. E., Clemens, M.J., and Kerr, I. M. (1976) Nature 264,477-480 K., and Krebs, E. G. (1987) 17. Edelman, A. M., Blumenthal, D.
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