Poliovirus protease does not mediate cleavage of the 220,000-Da

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1 Proc. Nati. Acad. Sci. USA Vol. 82, pp , May 1985 iochemistry Poliovirus protease does not mediate cleavage of the 22,-Da component of the cap binding protein complex (protein synthesis initiation factors/in vitro translation) RICHARD E. LLOYD*, DIANE ETCHISONt, AND ELLIE EHRENFELD*t *Departments of Cellular, Viral and Molecular iology and tiochemistry, University of Utah Medical Center, Salt Lake City, UT 84132; and tdepartment of iological Chemistry, School of Medicine, University of California, Davis, CA Communicated by Sidney F. Velick, January 14, 1985 ASTRACT Poliovirus infection of HeLa cells results in a rapid shutoff of host protein synthesis but does not inhibit the translation of poliovirus mrna. It has been suggested that this virus-induced translational control is mediated by the inactivation of a cap binding protein (CP) complex, and it has been shown that the 22,-Da component(s) (p22) of the CP complex is cleaved in infected HeLa cells to form antigenically related peptides of 1,-13, Da. To determine whether the known viral protease (peptide 3C) was the mediator of the cleavage of p22, we used immunoblot techniques to analyze partially purified infected HeLa cell extracts for cleavage activity. We report here that p22 cleavage activity does not copurify with viral peptide 3C or with any precursors containing 3C sequences. We also show that cleavage of p22 can be demonstrated in vitro in HeLa cell extracts under conditions where the functional activity of the poliovirus protease is inhibited by specific antibody. Poliovirus infection of HeLa cells induces a rapid and complete inhibition of host cell protein synthesis (1). Experiments with fractionated extracts from poliovirus-infected HeLa ceils have shown that the ribosomal salt wash (RSW) from infected cells, which contains protein synthesis initiation factors, did not support translation of host or other capped mrnas in vitro but did stimulate translation of poliovirus mrna (2-4). All other infected cell fractions did function in the translation of capped mrnas in this system. Thus, of the components of the cellular translational machinery, only initiation factors seem to be altered by poliovirus infection. Purification and analysis of individual initiation factors has implicated the cap binding protein (CP) complex (also called CP II or eif-4f) as the defective component in poliovirusinfected cells (5-8). This multisubunit protein is required for formation of ribosome initiation complexes with capped mrnas. Presumably, poliovirus mrna, which is not capped, initiates translation via a cap-independent mechanism. A CP complex has been purified from rabbit reticulocytes by workers in three laboratories, and it has been isolated as a complex of at least three polypeptides of 24,, 46,-5,, and 2,-22, Da (5-7). The smallest component of this complex (24, Da) was specifically crosslinked to the labeled cap group of oxidized mrna, which confirmed its identity with the 24,-Da cap binding protein (CP I) described previously (5, 6, 9). The 46,-Da component is eif-4a or a variant of eif-4a (6, 7). The largest component of the CP complex (termed p22) appears to be required for activity, but its precise function has not been characterized. Purified CP complex can restore translation of capped mrnas in poliovirus-infected cell extracts (5-7), The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. providing strong evidence that only CP complex is altered by virus infection. y use of a specific [3H]m7Gppp mrna crosslinking assay, Hansen et al. (1) showed that the 24,-Da CP I component of CP complex is found predominantly in a rapidly sedimenting form in uninfected cells and only in a slowly sedimenting form in poliovirus-infected cells, suggesting that virus infection leads to the disruption of the protein complex. Etchison et al. (11) used antiserum prepared against a preparation of eif-3 that also contained CP complex. Immunoblot analysis of HeLa cell proteins with this reagent showed that p22 could readily be detected in uninfected cells, but p22 was not present in infected cells. Instead, other antigenically related peptides of 1,-13, Da were detected. Presumably, these smaller peptides represent cleavage products of p22 and these data support the hypothesis that CP complex is defective in infected cells. These authors also reported a temporal correlation between the inhibition of host protein synthesis and the degradation of p22 in infected HeLa cells. Therefore, specific cleavage of p22 was suggested as the cause of the dissociation and inactivation of CP in poliovirus-infected cells. The poliovirus genome is known to code for at least one protease involved in processing precursor polypeptides to functional viral proteins (12). Antiserum to protein 3C [nomenclature according to Rueckert and Wimmer (13); formerly called P3-7c] specifically inhibited processing at Gln- Gly residues in vitro, and thus the activity responsible for these cleavages likely resides in the amino acid sequences contained in 3C (12). The antiserum also reacts with 3CD (P3-2) and 3C' (P3-6a), two viral polypeptides NH2- coterminal with 3C, and it is not known how many or which of these polypeptides manifest protease activity. The observation that the cellular protein p22, involved in capdependent translation, is proteolytically cleaved in poliovirus-infected cells has led to the suggestion that the viral protease 3C might be responsible for the cleavage and thus might be the direct viral mediator of the virus-induced inhibition of host cell protein synthesis (14). In this study, we tested this hypothesis, and we report that the viral protease 3C does not copurify with the p22 cleavage activity. We also demonstrate that the in vitro cleavage of p22 occurs under conditions in which the functional activity of the poliovirus protease is inhibited by specific antibody. MATERIALS AND METHODS Cells and Virus. HeLa S3 cells were grown in spinner cultures in Joklik's modified minimum essential medium supplemented with 7% calf serum. The growth and purifica- Abbreviations: CP complex, cap binding protein complex; RSW, ribosomal salt wash. 2723

2 2724 iochemistry: Lloyd et al. tion of poliovirus type 1 (Mahoney strain) has been described (15). Preparation of Cell Extracts and RSW. Approximately 2 x 19 HeLa cells were infected with poliovirus at a multiplicity of infection of 1 plaque-forming units (pfu) per cell, and the virus was adsorbed for 3 min at 37TC before serum was added to 2%. The cells were incubated for 4.5 hr before harvesting and extraction. Poliovirus proteins were specifically labeled by removing a fraction of the infected cell culture (5 x 17 cells) at 3.5 hr postinfection and for the remaining hour of incubation, by adding 1 mci of [35S]methionine (New England Nuclear; 15 Ci/mmol; 1 Ci = 37 Gq). At 4.5 hr postinfection, these cells were combined with nonlabeled infected cells and harvested. The cells were disrupted by Dounce homogenization, and postmitochondrial cytoplasmic extracts and RSW were obtained as described (15). RSW were obtained from mock-infected HeLa cells by the same protocol. Partial Purification of p22 Cleavage Activity. Infected cell RSW was precipitated wtih 4%o ammonium sulfate as described (16). The precipitate was resuspended in buffer A [2 mm TrisHCl, ph 7.4/5% (vol/vol) glycerol/7 mm 2-mercaptoethanol] containing 1 mm KCl and was dialyzed against the same buffer. This material was then applied to a DE-52 column (Whatman; 1.5 x 7 cm), and fractions were eluted with buffer A containing 1 mm KCl, 1 mm KCl, and then a continuous gradient of 1-5 mm KCl. Fractions containing p22 cleavage activity were pooled and dialyzed against 5 mm potassium phosphate, ph 7./5% (vol/vol) glycerol/7 mm 2-mercaptoethanol/1 mm KCl. The protein sample was applied to a P-1l phosphocellulose (Whatman) column (1.5 x 4 cm) and eluted in steps with the same buffer containing 1 mm KCl, 1 mm KCl, and 5 mm KCL. The eluted fractions were dialyzed against the starting phosphate buffer (1 mm KCl) before assay and analysis. Antibody and Imnmunoblot Analysis. Monoclonal antibody directed against p22 was isolated from a hybridoma that was prepared as a mouse ascites tumor. The antibody is a murine IgG1 with K light chains. It was developed against a crude CP complex preparation, and its characterization will be reported elsewhere. For immunoblotting, proteins were separated by NaDodSO4/polyacrylamide gel electrophoresis (17) and then transferred to nitrocellulose (Schleicher & Schuell, A 83) paper by electroblotting according to the procedure of urnette (18). To visualize p22, the nitrocellulose sheets were treated sequentially with anti-p22 monoclonal antibody, rabbit anti-mouse IgG, IgA, and IgM antiserum (Caliochem) and 1251-labeled staphylococcal protein A (Sigma). Radioiodination of protein A was carried out with lodogen beads (Pierce). Monoclonal antibody, rabbit antiserum, or protein A were suspended at >1:1 dilution in wash solution (5% nonfat dry milk,.5 M NaCl, 1 mm Tris HCl, ph 7.4) before use. The induction of rabbit anti-3c antiserum, kindly supplied by. Semler, has been described (12). IgG was prepared from this antiserum and from control rabbit antiserum by adsorption to a staphylococcal protein A- Sepharose (Pharmacia) column (19). The eluted IgG fraction was concentrated and dialyzed against buffer A with.3 M KCl before addition to in vitro translation reactions. In Vitro Translation. Endogenous mrna contained in cytoplasmic extracts from uninfected or infected cells was translated in vitro. Reaction mixtures (33 1d) contained 1 pl of cell lysate, [35S]methionine (25 uci, 15 Ci/mmol), 1 mm magnesium acetate, 9 mm potassium acetate, 2 mm Hepes (ph 7.6),.75 mm spermidine, 2 mm dithiothreitol, 25 mm creatine phosphate, 12 units of creatine phosphokinase, and 1.5 mm GTP. Proc. NatL. Acad. Sci. USA 82 (1985) RESULTS p22 Cleavage Assay. The monoclonal antibody used in this study to detect the 22,-Da component of the CP complex was developed against a partially purified CP complex. Fig. la shows that the antibody binds specifically to the p22 component of a purified preparation of CP complex (lane a). This CP complex preparation stimulates translation of capped globin mrna in the presence of initiation factors from poliovirus-infected cells and, thus, has functional CP complex activity (8). In addition, the antibody reacts only with the corresponding p22 material present in a complete cytoplasmic extract of uninfected HeLa cells (Fig. LA, lane b). As expected, this antigen is present in the RSW from uninfected cells (Fig. 1, lane c), but no p22 is detectable in RSW from polio-infected cells. As previously reported, smaller presumed cleavage products of p22 (1,-13, Da) are identified by the antibody in the infected cell RSW (Fig. 1, lane d). p22 has usually been described as a single polypeptide; however, when analyzed on gels of low percentage acrylamide, up to four polypeptide moieties can be detected with the monoclonal antibody. The immunoblots of infected cell extracts also reveal multiple peptides with common antigenic specificity, with two peptides being major species. Purified CP complex from HeLa cells also yields multiple p22 protein bands by Coomassie blue protein staining (data not shown). Therefore, the p22 component ofcp complex appears to exist in several forms, with slightly different mobilities in polyacrylamide gels. Since all the larger peptides are cleaved in infected cells (Fig. 1), for the purposes of discussion in this manuscript, they will be collectively referred to as p22. It has been shown previously that RSW preparations from polio-infected HeLa cells contain an activity that will cleave x a) E - ca Cl a (o) u) u b cr c Cd FIG. 1. Immunoautoradiographic identification of p22 and p22 cleavage products in extracts from uninfected and infected cells. HeLa cytoplasmic extract, RSW, or purified CP complex was electrophoretically transferred from 1% NaDodSO4/polyacrylamide gels to nitrocellulose and treated with anti-p22 and 1"-I-labeled protein A. Samples analyzed were DEAE-purified CP complex (lane a), cytoplasmic extract from uninfected cells (lane b), RSW from uninfected cells (lane c), and RSW from poliovirusinfected cells (lane d). Autoradiography was performed for 18 hr.

3 iochemistry: Lloyd et al. exogenous p22 present in RSW prepared from uninfected cells (11). We have used the immunoblot procedure to assay fractions from infected cells for p22 cleavage activity. Fig. 2 (lanes c-f) shows that infected cell RSW rapidly converts all of the p22 present in an uninfected cell RSW to the characteristic smaller products seen in infected cells. No such activity is present in the corresponding fraction from uninfected cell RSW (lane b). Poliovirus Protease (Peptide 3C) Does Not Copurify with p22 Cleavage Activity. The immunoblot assay described above was used to localize p22 cleavage activity during initial purification protocols. At the same time, samples were analyzed for the presence of poliovirus-specific polypeptides that were specifically radiolabeled during infection prior to preparation of the RSW. In particular, it was of interest to determine whether viral protein 3C, the virus-encoded protease (12, 14), co-purified with p22 cleavage activity. RSW from infected cells was fractionally precipitated with ammonium sulfate into two fractions (%-4% saturation, called fraction A; 4%-7% saturation, called fraction ). Immunoblot assay showed fraction A to contain the majority (>9%) of the p22 cleavage activity (data not shown). Therefore, this material was further fractionated by ionexchange chromatography on DEAE-cellulose. All fractions were then analyzed by the immunoblot assay for p22 cleavage activity. Fig. 3A shows that the cleavage activity was detected in the whole cell cytoplasmic extract, the ammonium sulfate fraction A, and in fractions eluting at M KCl (DEAE fractions 4, 5, and 6; lanes h-j). This represents the leading edge of the major peak of total protein that bound to the column (data not shown). Fig. 3 shows the location of poliovirus-specific polypeptides in these fractions and demonstrates that the viral protease, peptide 3C, bound the column only wealdy at.1 M salt (lane c) and was completely removed by the.1 M salt wash (lane d). No [35S]methionine-labeled protease peptide was detected in fractions 4, 5, and 6, which contained p22 cleavage activity (lanes h-j). a b c d e f g U-RSW I-RSW inc FIG. 2. Immunoautoradiographic assay for p22 cleavage activity. RSW (5 /) from uninfected cells was incubated alone for 6 min at 37 C (lane b) or with RSW (5 pl) from poliovirus-infected cells for min (lane c), 15 min (lane d), 3 min (lane e), or 6 min (lane f) at 37C. RSW from uninfected cells (lane a) or infected cells (lane g) was also examined. All samples were analyzed by l1o NaDodSO4/ polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose. p22 and related peptides were detected by treatment with p22-specific antibody and 125I-labeled protein A and autoradiography for 18 hr. A Proc. Natl. Acad. Sri FJS4. 82 (1985) 2725 C 3CD--- 3D- - DEAE Fractions _ - -, > Uco l o bcdef ghi j k Imn a bc d e f g h j k m n VPO - VPI- - VP2.-- VP3-3C - VP4- FIG. 3. Separation of p22 cleavage activity from poliovirus protease 3C by DEAE column chromatography. RSW from infected cells was precipitated with ammonium sulfate and fraction A was applied to DEAE. (A) Column fractions were incubated with 5 p1 of RSW from uninfected cells at 37 C for 2 hr and analyzed for p22 and related peptides as described in the legend to Fig. 2. Fractions assayed were 5 A1 of cytoplasmic extract of infected cells (lane a), 5 Ad of fraction A (lane b), 2 p1 of column flowthrough (lane c), 2,ul of.1 M KCI eluate (lane d), 2 Ad of sequential fractions eluted by a continuous.1-.5 M KCI gradient (lanes e-n). Also shown is 5 Ad of RSW incubated alone (left lane). () DEAE column fractions were analyzed by 12.5% NaDodSO4/polyacrylamide gel electrophoresis and autoradiography for 35S-labeled poliovirus proteins. Virion proteins are shown and all other lanes (a-n) are the same as described above. Separation of p22 Cleavage Products From the Cleavage Activity. The infected cell RSW used as starting material for the preliminary purification of p22 cleavage activity also contains the pre-existing products of the cleavage reaction that were present in the infected cells. In fact, immunoblot analysis of DEAE column fractions indicated that these antigenically reactive products of 1,-13, Da copurifled with the cleavage activity through DEAE chromatography (data not shown). However, when the pooled fractions from the DEAE column were applied to a phosphocellulose column under low salt conditions, p22 cleavage activity failed to bind and was found in the column flow-through fraction (Fig. 4, lane g). p22 cleavage products, however, do bind to the resin under these conditions, and they are only eluted when the KCl concentration is increased to.1 M (lane d). Resolution of the endogenous cleavage products from the cleavage activity improves the quality of the cleavage assay since both appearance of product as well as disappearance of substrate can be seen on the immunoblots. Absence of Viral Protease Sequences in p22 Cleavage Fractions. Amino acid sequences in viral protein 3C have

4 2726 iochemistry: Lloyd et al. Proc. NatL. Acad. Sci. USA 82 (1985) ( D _ o E :E L.LJ z <r - o LLJ ) CO :: cl cr x Z) 7DD D - u -_ y y CAL 2i llx Y.E E~ w _ E E - In < dw - un O r-66 o polio proteins 3CD- Lii ;J, %.: dti v - C Qa m,~ammim VPO\ 3C VPI- VP2- VP3 3C- a bc d e f g h i FIG. 4. Separation of p22 cleavage activity from cleavage products by column chromatography. Pooled fractions 4, 5, and 6 from the DEAE column were chromatographed on phosphocellulose. Column fractions and RSW from uninfected cells were incubated alone or together for 4 hr at 37C and were analyzed for p22 as described in Fig. 2. Fractions were 3 p4 of RSW from uninfected cells (lane a), 2 ja of pooled DEAE fractions incubated alone (lane b) or with RSW (lane f), P-11 flowthrough incubated alone (lane c) or with RSW (lane g), 2,ul of P-11.1 M KCI eluate incubated alone (lane d) or with RSW (lane h), and 2 pj of P-11.5 M KCI eluate incubated alone (lane e) or with RSW (lane i). been demonstrated to be responsible for the cleavage of poliovirus protein precursors at Gln-Gly residues (12). Protein 3C is itself processed, perhaps autocatalytically (14), from precursor proteins 3ACD (formerly P3-lb) and 3CD (formerly P3-2). In addition, protein 3CD can undergo an alternative cleavage pathway to generate two polypeptides, one of which contains 3C sequences, of no known function. Since it has not been conclusively demonstrated which of these proteins, all of which contain 3C amino acid sequences, actually catalyzes proteolytic reactions, it was important to determine whether p22 cleavage activity was associated with any proteins comprised of viral 3C sequences. Samples were resolved by NaDodSO4/polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and then underwent reaction with antiserum raised against protein 3C (12). Fig. 5 shows that the antiserum reacts with 3C and precursor protein 3CD, as well as with the alternate cleavage product (protein 3C', formerly P3-6a) in poliovirus-infected HeLa cytoplasmic extracts (lane a), and in the ammonium sulfate fraction A (lane b). The DEAE eluate contained no detectable 3C, although some precursor proteins are present (lane c), but no reactive antigens remain in the phosphocellulose flow-through (lane d), which contained peak p22 cleavage activity. The antiserum reacts with two polypeptides (22, and 2, Da) present in infected cytoplasmic extracts (lanes a and b), the smaller of which comigrates with polypeptide 3C. The larger of these two bands might be a cellular polypeptide that contaminated the immunogen used to raise the antiserum, or it might be a slightly larger precursor to 3C, with additional NH2-terminal viral protein sequences. a b c d FIG. 5. Immunoautoradiographic detection of poliovirus protease (3C). Samples of infected cell lysates and purified fractions that contain p22 cleavage activity were analyzed for viral protease sequences by 12.5% NaDodSO4/polyacrylamide gel electrophoresis and electroblotting to nitrocellulose. Nitrocellulose was then treated with anti-3c antiserum and "MI-labeled protein A as described. Fractions analyzed were 5 pd of infected cell cytoplasmic extract (lane a), 5 pl of fraction A (lane b), 2 /4 of pooled fractions 4, 5, and 6 from DEAE chromatography (lane c), and 2 y4 of flowthrough fraction from P11 column chromatography (lane d). Effect of Anti-3C IgG on Cleavage of p22 in Vitro. Antibodies from the anti-3c antiserum described above have been shown to inhibit processing of poliovirus precursor proteins synthesized in vitro in an extract from infected HeLa cells (12). Cleavages at Gln-Gly pairs were specifically inhibited by anti-3c IgG, whereas other cleavages known to occur at Tyr-Gly pairs were unaffected. An infected cell extract was prepared and incubated under protein synthesis conditions in vitro. The synthesis and processing of poliovirus proteins by this extract is shown in Fig. 6A (lane a). When the extract was preincubated with IgG purified from anti-3c antiserum, the pattern of synthesized proteins changed markedly (Fig. 6A, lanes d and e). The majority of synthesized protein remained large, and the appearance of almost all cleavage products was inhibited (e.g., see arrows in Fig. 6A indicating cleavage products 2C and 2C). Of notable exception is polypeptide 3D', whose formation appears to be enhanced; this polypeptide results from a Tyr-Gly cleavage, not inhibited by anti-3c IgG (12). A small amount of RSW that contained intact p22 from uninfected HeLa cells was included in the translation reactions. Fig. 6 (lanes d and e) shows that cleavage of p22 occurred completely in the same reaction in which cleavage of poliovirus precursor protein was inhibited. This result further demonstrates that cleavage of p22 is not directly affected by the viral protease that processes viral proteins. DISCUSSION Numerous lines of evidence suggest that the poliovirusinduced inhibition of host cell protein synthesis results from inactivation of the CP complex activity. The structural basis for this inactivation is still under investigation. It has been shown that the rapidly sedimenting complex that contains

5 iochemistry: Lloyd et al A a b c d e f M V a b c d e f R FIG. 6. Effect of anti-3c IgG on in vitro processing of poliovirus proteins and p22 cleavage activity. Purified IgG was preincubated on ice for 6 min with 1 1J of cytoplasmic extracts from poliovirusinfected cells. Translation reaction mix (9 IL) containing [35S]methionine and 5 pa of RSW from uninfected cells was added, and the reactions were incubated 9 min at 37TC. (A) Effect ofanti-3c IgG on poliovirus protein processing. Samples (15 p1) from the in vitro translation reactions were analyzed by 1% NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Translation reactions analyzed contained infected lysate incubated without IgG (lane a), with 23 Mg of normal rabbit IgG (lane b), with 23 jig of normal rabbit IgG (lane c), with 16 jg of anti-3c IgG (lane d), with 16 Mg of anti-3c IgG (lane e), or uninfected cell lysate alone (lane f). In vivo labeled poliovirus marker proteins (lane M) and labeled virion capsid proteins (lane V) are also shown. () Effect of anti-3c IgG on p22 cleavage in vitro. Samples (15 pa) of translation reactions were analyzed for p22 and p22 breakdown products as described in the legend to Fig. 2. Translation reactions electroblotted (lanes a-f) were the same as described above. RSW from uninfected cells (5 pi) is shown in lane R. Proc. Nati. Acad. Sci. USA 82 (1985) ,-Da CP I is dissociated, or unstable to standard purification procedures, in infected cells (1); it has also been demonstrated that the p22 component of the CP complex is cleaved in infected cells (11). If proteolysis of p22 is the direct event responsible for inactivation of cap-dependent protein synthesis after poliovirus infection, it seemed reasonable to test the hypothesis that the known poliovirus-encoded protease was the mediator of p22 cleavage. The cleavage site specificity of the viral protease appears to involve stringent recognition of Gln-Gly pairs in the substrate. The site(s) of cleavage in p22 have not been identified. We have used an immunoblot technique to detect the cleavage of p22 in vitro, by fractions from poliovirusinfected HeLa cell lysates. This assay enabled us to partially purify the cleavage activity in infected cells and to demonstrate that the cleavage activity separates from radiolabeled viral protease 3C at an early step in purification. Immunoblotting with specific antibody against the viral protease corroborated these data and also eliminated the possibility that protease precursors (3CD) or related polypeptides (3C') were alternative mediators of p22 cleavage. Fractions that were markedly enriched for viral protease 3C had no detectable p22 cleavage activity. Although it was not possible to demonstrate that these fractions were active on poliovirus precursor proteins because of unavailability of substrate, it was possible to inhibit the action of the viral protease in an infected cell translation system with IgG prepared from anti-3c antiserum. Under these conditions, cleavage of p22 was not affected. Thus, by this criterion as well, viral 3C sequences are not involved in the cleavage of the p22 component of the CP complex. The identity of the mediator of p22 cleavage remains in question. No major poliovirus proteins clearly copurify with the cleavage activity (data not shown), but neither a weakly labeled viral protein nor a cellular protein that is altered or activated by poliovirus infection can be excluded at this time. If the latter is the case, the viral protease could play a role in the activation and, thus, might be indirectly involved in the translational regulation observed in infected cells. Despite the relatively small size of the poliovirus genome, only the P1 and P3 regions, representing the 5' and 3' segments of the coding sequence, have been assigned gene products with known functions. Proteins encoded by the middle region have no identified functions and, thus, can still be considered candidates for p22 cleavage activity. We are grateful to Dr. R. Hanecak, Dr.. Semler, and Dr. E. Wimmer for furnishing antibody to peptide 3C and for helpful discussions, and to J. Cohenour for preparation of the manuscript. This work was supported by Public Health Service Grants AI (E.E.), AI (D.E.), and Fellowship AI-785 (R.E.L.). 1. Ehrenfeld, E. (1982) Cell 28, Kaufmann, Y., Goldstein, E. & Penman, S. (1976) Proc. NatI. Acad. Sci. USA 73, Helentjaris, T. & Ehrenfeld, E. (1978) J. Virol. 26, rown, D., Hansen, J. & Ehrenfeld, E. (198) Virology 13, Tahara, S. M., Morgan, M. A. & Shatkin, A. J. (1981) J. iol. Chem. 256, Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J. & Merrick, W. C. (1983) J. iol. Chem. 258, Edery, I., Humbelin, M., Darveau, A., Lee, K., Milburn, S., Hershey, J., Trachsel, H. & Sonenberg, N. (1983) J. iol. Chem. 258, Etchison, D., Hansen, J., Ehrenfeld, E., Edery, I., Sonenberg, N., Milburn, S. & Hershey, J. W.. (1984) J. Virol. 51, Sonenberg, N., Morgan, M., Merrick, W. & Shatkin, A. J. (1978) Proc. Natl. Acad. Sci. USA 75, Hansen, J. L., Etchison, D., Hershey, J. W.. & Ehrenfeld, E. (1982) J. Virol. 42, Etchison, D., Milburn, S. C., Edery, I., Sonenberg, N. & Hershey, J. W.. (1982) J. iol. Chem. 257, Hanecak, R., Semler,. L., Anderson, C. W. & Wimmer, E. (1982) Proc. Natl. Acad. Sci. USA 79, Rueckert, R. R. & Wimmer, E. (1984) J. Virol. 5, Hanecak, R., Semler,. L., Ariga, H., Anderson, C. W. & Wimmer, E. (1984) Cell 37, Jones, C. & Ehrenfeld, E. (1983) Virology 129, rown,. A. & Ehrenfeld, E. (198) Virology 13, Laemmli, U. K. (197) Nature (London) 227, urnette, W. N. (1981) Anal. iochem. 112, Oi, V.T. & Herzenberg, L. A. (198) in Selected Methods in Cellular Immunology, eds. Mishell,.. & Shiigi, S. M. (Freeman, San Francisco), pp

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